JP2016528288A - Life prolongation effect in patients with solid tumors with elevated C-reactive protein levels - Google Patents

Abstract

The present application relates to the administration of a JAK inhibitor or an IL-6 signaling inhibitor to a patient, wherein the patient has a solid tumor, where the patient has an elevated serum concentration of C-reactive protein (CRP). It relates to methods for prolonging survival or progression-free survival, and methods for predicting life-prolonging effects in these patients derived from such therapy.

Description

  This application claims the benefit of priority of US Provisional Application No. 61 / 867,982, filed Aug. 20, 2013, which is hereby incorporated by reference in its entirety.

  The present application relates to the survival of solid tumor patients (where the patient has an elevated serum concentration of C-reactive protein (CRP)) by administering to the patient a JAK inhibitor or an IL-6 signaling inhibitor. Or, it relates to a method for prolonging progression-free survival and a method for predicting the survival benefit in these patients derived from such therapy.

Janus kinase (JAK) plays an important role in signaling after cytokines and growth factors bind to their receptors. Abnormal activation of JAK through abnormal or excessive cytokine signaling or through intracellular mechanisms that cause pathway dysregulation has been thought to be associated with increased malignant cell proliferation and increased survival.
There are multiple mechanisms by which inflammatory cytokines can affect tumor growth and survival. Cytokines are important molecules that control autocrine and paracrine communication within and between tumor cells and between their surrounding stromal environment and tumor cells. Under some conditions, endogenous cytokines can organize host responses to tumors, and the cytokine network also contributes to tumor growth, progression and host immune suppression. In addition, inflammatory cytokines have been shown to be important mediators of catabolic and cachexia associated with cancer, and thus inflammatory cytokines are mediated through this mechanism and direct effects on tumor cells. Can affect the course of cancer patients. C-reactive protein (CRP) is a protein that can be measured in serum, is a major indicator of systemic inflammatory response, and is associated with elevated levels of cytokines, particularly IL-6. Elevated CRP was associated with poor prognosis and low responsiveness to conventional therapy in a wide range of tumors (Non-Patent Document 1). There is a medical need to improve the treatment of cancer patients with this poor prognostic factor. The present invention is directed to this and other needs.

McMillan, D.M. C. , Cancer Treatment Reviews 39 (2013) 534-540

  The present application provides a method of extending survival or progression-free survival, particularly in solid tumor patients, wherein the patient has an elevated serum concentration of C-reactive protein (CRP), the method comprising: Administration of a Janus kinase (JAK) inhibitor or IL-6 signaling inhibitor to the patient, wherein the administration extends the survival or progression-free survival of the patient.

  The application also provides a method for prolonging survival or progression-free survival in a solid tumor patient, wherein the patient has an improved Glasgow Prognostic Score (mGPS) of 1 or 2 And the method comprises administering a Janus kinase (JAK) inhibitor or IL-6 signaling inhibitor to the patient, wherein the administration prolongs the survival or progression-free survival of the patient. .

The application further provides a method of treating a solid tumor in a patient in need of the method, wherein the patient has an improved Glasgow Prognostic Score (mGPS) of 1 or 2, the method comprising: Administering a Janus kinase (JAK) inhibitor or an IL-6 signaling inhibitor to the patient. In addition, this application:
(A) selecting a solid tumor patient having a serum concentration of C-reactive protein (CRP) that is greater than or equal to the median CRP baseline serum concentration for a patient population having a solid tumor;
(B) providing a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

In addition, this application:
(A) selecting a solid tumor patient having a serum concentration of C-reactive protein (CRP) of about 10 μg / mL or greater;
(B) providing a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

In addition, this application:
(A) selecting solid tumor patients with an improved Glasgow prognostic score of 1 or 2;
(B) providing a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

  The application also provides a method for predicting the efficacy of treatment using a JAK inhibitor or an IL-6 signaling inhibitor for a patient with a solid tumor, the method comprising the patient's C-reactive protein Comparing the serum concentration of (CRP) to the CRP baseline serum concentration of the solid tumor patient population, wherein the serum CRP concentration of the patient above the baseline serum concentration is a JAK inhibitor or IL-6 signaling Represents the effectiveness of a treatment using an inhibitor for a patient.

  In addition, this application also provides JAK inhibitors or IL-6 signaling inhibitors for use described in any of the methods described by the embodiments described herein.

  The application provides for the use of a JAK inhibitor or an IL-6 signaling inhibitor to prepare a medicament for use in any of the methods described by the embodiments described herein.

Shown is Kaplan-Meier analysis of overall survival for patients whose baseline CRP was 13 μg / mL or less (survival distribution function versus days for groups 1 and 2). Shown is Kaplan-Meier analysis of overall survival for patients with baseline CRP> 13 μg / mL (survival distribution function versus days for groups 1 and 2). FIG. 6 shows Kaplan-Meier analysis of progression-free survival for patients with baseline CRP of 13 μg / mL or less (survival distribution function versus days for groups 1 and 2). Shows Kaplan-Meier analysis of progression-free survival for patients with baseline CRP> 13 μg / mL (survival distribution function versus days for groups 1 and 2). (A)-(C): shows Kaplan-Meier curves for overall survival by baseline mGPS (A, mGPS = 0; B, mGPS = 1; and C, mGPS = 2), (y-axis is survival distribution function) Yes; y-axis is days of survival).

DETAILED DESCRIPTION This application provides a method for prolonging survival or progression-free survival in a solid tumor patient, wherein the patient has an elevated serum concentration of C-reactive protein (CRP), the method comprising: Administering a JAK inhibitor or IL-6 signaling inhibitor to the patient, wherein the administration prolongs the patient's survival or progression-free survival.

  In some embodiments, the method further comprises selecting a patient having an elevated serum concentration of C-reactive protein prior to administration.

  In some embodiments, the elevated serum concentration of CRP is a serum concentration that is greater than or equal to the median CRP baseline serum concentration for a patient population with a solid tumor (ie, as measured by a CRP assay).

  In some embodiments, the elevated serum concentration of CRP is that of about 10 μg / mL or greater.

  In some embodiments, the elevated serum concentration of CRP is that of more than twice the upper limit of normal.

  In some embodiments, the elevated serum concentration of CRP is that of at least 2.5 times the upper limit of normal.

  In some embodiments, the elevated serum concentration of CRP is that of more than 3 times the upper limit of normal.

  In some embodiments, the elevated serum concentration of CRP is that of at least 3.5 times the upper limit of normal.

  In some embodiments, the elevated serum concentration of CRP is that of more than 4 times the upper limit of normal.

In addition, this application:
(A) selecting a solid tumor patient having a serum concentration of C-reactive protein (CRP) that is greater than or equal to the median CRP baseline serum concentration for a patient population having a solid tumor;
(B) providing a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

In addition, this application:
(A) selecting a solid tumor patient having a serum concentration of C-reactive protein (CRP) that is about 10 μg / mL or greater;
(B) There is also provided a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

  In some embodiments, the administration extends patient survival.

  In some embodiments, the administration extends the patient's progression-free survival.

  In some embodiments, the serum concentration of CRP is about 13 μg / mL or greater.

  In some embodiments, the serum concentration of CRP is about 10 μg / mL or greater.

In some embodiments, this application:
(A) selecting patients with solid tumors with an improved Glasgow prognostic score of 1 or 2;
(B) providing a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

  In some embodiments, the administration extends patient survival.

  In some embodiments, the administration extends the patient's progression-free survival.

血清ＣＲＰ濃度は、標準的な市販のアッセイまたはＲｕｌｅｓ Ｂａｓｅｄ Ｍｅｄｉｃｉｎｅｓ（ＲＢＭ）アッセイを使用して測定することができる。ＣＲＰのための市販の臨床アッセイとしては、Ｑｕｅｓｔ Ｄｉａｇｎｏｓｔｉｃｓ Ｃ−Ｒｅａｃｔｉｖｅ Ｐｒｏｔｅｉｎ（ＣＲＰ）試験またはＬａｂｃｏｒｐ ｃ−Ｒｅａｃｔｉｖｅ Ｐｒｏｔｅｉｎ Ｈｉｇｈ Ｓｅｎｓｉｔｉｖｉｔｙ試験が挙げられるが、それらに限定されない。ＲＢＭアッセイとしては、ＲＢＭ多重化Ｌｕｍｉｎｅｘ（登録商標）市販アッセイ（Ｍｙｒｉａｄ ＲＢＭ）が挙げられるが、それらに限定されない。市販の臨床アッセイは相関され得る。例えば、ＲＢＭアッセイにおける１０μｇ／ｍＬの血清濃度は、臨床アッセイにおける約１０μｇ／ｍＬと相関していると考えられる。 An improved version of Glasgow Prognostic Score (mGPS) is described in McMillian, Cancer Treatment Reviews, 39 (5): 534-540 (2013), which is incorporated herein by reference in its entirety (see Table 1 for details). Score, duplicated below).

Serum CRP concentrations can be measured using standard commercial assays or the Rules Based Medicines (RBM) assay. Commercial clinical assays for CRP include, but are not limited to, the Quest Diagnostics C-Reactive Protein (CRP) test or the Labcorp c-Reactive Protein High Sensitivity test. RBM assays include, but are not limited to, RBM multiplexed Luminex® commercial assay (Myriad RBM). Commercial clinical assays can be correlated. For example, a serum concentration of 10 μg / mL in the RBM assay is thought to correlate with about 10 μg / mL in the clinical assay.

  CRP tests are approved by the FDA under the 510K process, and most of the available tests are predicate tests that have established criteria for analytical validation of individual tests as well as the analytical platform where the tests are conducted. A test substantially equivalent to 510K based on test) was utilized. Conventional CRP assays are common indicators used to assess infection, tissue damage, and inflammatory disorders. These assays provide information for the diagnosis, therapy and monitoring of inflammatory diseases (FDA Guidance for Industry-Review Criteria for Assessment of C Reactive Protein (CRP), High Sensitive C-Reactivity C-P C-Reactive Protein (cCRP) Assays, www.fda.gov/medicaldevices/deviceregulationandguidance/guidancements/ucm077167.htm (accessed on September 17, 2011). CRP is one of the “acute phase” proteins induced by cytokines, and its blood levels are elevated during a general, nonspecific response to infectious and non-infectious inflammatory processes (Peys and Hirschfield). , J Clin Invest 2003 111: 1805-1812). CRP reflects ongoing inflammation and / or tissue damage more accurately than other laboratory parameters of the acute phase response, such as plasma viscosity and erythrocyte sedimentation rate. Importantly, acute CRP values do not show circadian variation and are not affected by feeding. Liver failure impairs CRP production, but any other concomitant pathology and drugs rarely reduce CRP levels if they do not affect the underlying pathology that provides acute phase stimulation. Therefore, CRP concentration is a very useful non-specific biochemical marker for inflammation (Pepys and Hirschfield 2003). In conventional CRP assays, test values are typically considered clinically significant at levels above 10 mg / L (FDA CRP Guidance).

  The assessment of inflammation associated with cancer using CRP as part of mGPS is well established in the medical literature (McMillan, Cancer Treat Rev 2013; 39: 534-540) and is a conventional CRP assay. Within the scope of approved labeling and intended use. The separation value that distinguishes mGPS 0 from mGPS 1 and 2 is the same as that generally accepted as clinically significant. Eligibility determination using CRP as part of mGPS is performed in a central laboratory using a single FDA approved assay system for all test subjects.

  As used herein, the term “JAK inhibitor” is intended to mean a compound that inhibits at least JAK1 and / or JAK2. In some embodiments, the JAK inhibitor is a JAK2 inhibitor. In some embodiments, the JAK inhibitor is a JAK1 inhibitor.

  In some embodiments, a JAK inhibitor can also inhibit other members of the Janus kinase family (ie, JAK3 or TYK2). In some embodiments, the JAK inhibitor is selective. “Selective” means that the compound binds JAK1 and / or JAK2, respectively, with greater affinity or potency compared to at least one other JAK (eg, JAK2, JAK3 and / or TYK2), respectively. Mean to inhibit. In some embodiments, the JAK inhibitor is selective for JAK1 and JAK2 relative to JAK3 and TYK2. In some embodiments, the compounds of the invention are selective inhibitors of JAK1 relative to JAK2, JAK3 and TYK2. Selectivity can be at least about 5 times, at least about 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 500 times or at least about 1000 times. Selectivity can be measured by routine methods in the art. In some embodiments, selectivity can be tested with the Km of each enzyme. In some embodiments, the selectivity of a compound for JAK1 and / or JAK2 can be measured by cellular ATP concentration.

  In some embodiments, the method comprises administering a JAK1 and / or JAK2 inhibitor to the patient.

  In some embodiments, the method comprises administering a JAK1 inhibitor to the patient.

  In some embodiments, the method comprises administering a JAK2 inhibitor to the patient.

  In some embodiments, the method comprises administering an IL-6 signaling inhibitor to the patient.

  In some embodiments, the JAK inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.

  In some embodiments, the JAK inhibitor is ruxolitinib phosphate.

In some embodiments, the JAK inhibitor is a selective JAK1 inhibitor. As used herein, a “selective JAK1 inhibitor” is an inhibitor of JAK1 that is selective for JAK1 over JAK2, JAK3 and TYK2. In some embodiments, the compound or salt thereof is about 10 times more selective for JAK1 than JAK2. In some embodiments, the compound or salt thereof is selected from about 10-fold, about 15-fold, or about 20-fold for JAK1 as compared to JAK2, as calculated by measuring IC ₅₀ at 1 mM ATP. (See, eg, Example A).

＋ 平均＜１０ｎＭ（アッセイ条件については実施例Ａを参照されたい）
＋＋ 平均≦１００ｎＭ（アッセイ条件については実施例Ａを参照されたい）
＋＋＋ 平均≦３００ｎＭ（アッセイ条件については実施例Ａを参照されたい）
^ａ エナンチオマー１に関するデータ
^ｂ エナンチオマー２に関するデータ In some embodiments, the selective JAK1 inhibitor is a compound of Table A or a pharmaceutically acceptable salt thereof. The compounds in Table A are selective JAK1 inhibitors (selective compared to JAK2, JAK3, and TYK2). The IC ₅₀ obtained by the method of Assay A with 1 mM ATP is shown in Table A.

+ Mean <10 nM (see Example A for assay conditions)
++ Mean ≦ 100 nM (see Example A for assay conditions)
+++ Mean ≦ 300 nM (see Example A for assay conditions)
data related to ^a single enantiomer
^b Data on Enantiomer 2

  In some embodiments, the selective JAK1 inhibitor is {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo). [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof.

  In some embodiments, the selective JAK1 inhibitor is {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo). [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile adipate.

  In some embodiments, the selective JAK1 inhibitor is 4- {3- (cyanomethyl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol- 1-yl] azetidin-1-yl} -2,5-difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide or a pharmaceutically acceptable salt thereof. .

  In some embodiments, the selective JAK1 inhibitor is (R) -3- [1- (6-chloropyridin-2-yl) pyrrolidin-3-yl] -3- [4- (7H-pyrrolo [ 2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanenitrile, (R) -3- (1- [1,3] oxazolo [5,4-b] pyridin-2- Ylpyrrolidin-3-yl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanenitrile, (R) -4-[( 4- {3-cyano-2- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propyl} piperazin-1-yl) carbonyl] -3 -Fluorobenzonitrile, (R) -4-[(4- {3-cyano-2- [3 (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrrol-1-yl] propyl} piperazin-1-yl) carbonyl] -3-fluorobenzonitrile, or (R) -4- (4- {3-[(Dimethylamino) methyl] -5-fluorophenoxy} piperidin-1-yl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H -Pyrazol-1-yl] butanenitrile, (S) -3- [1- (6-chloropyridin-2-yl) pyrrolidin-3-yl] -3- [4- (7H-pyrrolo [2,3- d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanenitrile, (S) -3- (1- [1,3] oxazolo [5,4-b] pyridin-2-ylpyrrolidine-3 -Yl) -3- [4- (7H-pyrrolo [ , 3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propanenitrile, (S) -4-[(4- {3-cyano-2- [4- (7H-pyrrolo [2, 3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propyl} piperazin-1-yl) carbonyl] -3-fluorobenzonitrile, (S) -4-[(4- {3-cyano 2- [3- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrrol-1-yl] propyl} piperazin-1-yl) carbonyl] -3-fluorobenzonitrile, ( S) -4- (4- {3-[(Dimethylamino) methyl] -5-fluorophenoxy} piperidin-1-yl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidine-4 -Yl) -1H-pyrazol-1-yl] butane A nitrile; and a pharmaceutically acceptable salt of any of the foregoing compounds.

  In some embodiments, the compounds of Table 1 are disclosed in US Patent Publication No. 2010/0298334, filed May 21, 2010, US Patent Publication No. 2011/0059951, filed August 31, 2010. US Patent Publication No. 2011/0224190 filed on March 9, 2011; US Patent Publication No. 2012/0149682 filed on November 18, 2011; US filed on November 18, 2011 Patent Publication No. 2012/0149682, US Patent Publication No. 2013/0018034 filed on June 19, 2012, US Patent Publication No. 2013/0045963 filed on August 17, 2012, and May 2013 Prepared by the synthetic procedure described in 2014/0005166, filed on May 17, Each serial US patent publication entirely incorporated herein by reference.

  In some embodiments, the selective JAK1 inhibitor is US Patent Publication No. 2010/0298334 filed May 21, 2010, US Patent Publication No. 2011/0059951 filed August 31, 2010. US Patent Publication No. 2011/0224190 filed on March 9, 2011; US Patent Publication No. 2012/0149682 filed on November 18, 2011; US filed on November 18, 2011 Patent Publication No. 2012/0149682, US Patent Publication No. 2013/0018034 filed on June 19, 2012, US Patent Publication No. 2013/0045963 filed on August 17, 2012, and May 2013 Selected from the compounds of US Patent Publication No. 2014/0005166, filed on May 17, Each of U.S. Patent Publication entirely incorporated herein by reference.

  In some embodiments, the method comprises administering to the patient about 15 mg to about 25 mg BID ruxolitinib or a pharmaceutically acceptable salt thereof, based on the free base. In some embodiments, the method comprises administering to the patient about 10 mg to about 25 mg BID of ruxolitinib or a pharmaceutically acceptable salt thereof based on the free base. In some embodiments, the method comprises administering to the patient about 15 mg to about 25 mg QD ruxolitinib or a pharmaceutically acceptable salt thereof, based on the free base. In some embodiments, the method comprises administering to the patient about 10 mg to about 25 mg QD ruxolitinib or a pharmaceutically acceptable salt thereof based on the free base.

  In some embodiments, the JAK inhibitor is US 7,598,257, US 7,834,022, US 2009/0233903, US 2010/0298355, US 2011-0207754, US 2010-0298334, US 2011-0059951. US 2011-0224190, US 2012-0149682, US 2012-0149682, US 2013-0018034, US 2013-0045963, US Patent Application No. 13 / 896,802, filed May 17, 2013, November 2011. US Patent Application No. 61 / 721,308 filed on May 1 or US Patent Application No. 61 / 824,683 filed May 17, 2013 Each of its There are incorporated herein by reference.

  The application further provides a method for predicting the efficacy of treatment using a JAK inhibitor or an IL-6 signaling inhibitor for a patient with a solid tumor, the method comprising the patient's C-reactive protein Comparing the serum concentration of (CRP) to the baseline serum concentration of CRP in a patient population having a solid tumor, wherein the serum CRP concentration of a patient above the baseline serum concentration is a JAK inhibitor or IL- 6 represents the efficacy of a treatment using a 6 signaling inhibitor on a patient.

  The present application provides a method for predicting the efficacy of a treatment using ruxolitinib or a pharmaceutically acceptable salt thereof for a patient with pancreatic cancer, the method comprising: serum concentration of C-reactive protein (CRP) in the patient Is compared to the baseline serum concentration of CRP in a patient population with a solid tumor, wherein the serum CRP concentration of the patient above the baseline serum concentration is an inhibitor of ruxolitinib or a pharmaceutically acceptable salt thereof It represents the effectiveness of treatment using salt on patients. In some embodiments, the method further comprises measuring the serum concentration of the patient's CRP using a CRP assay prior to the comparison.

  In some embodiments, the predicting method further comprises measuring the serum concentration of the patient's CRP using a CRP assay prior to the comparison.

  In some embodiments, the predicting method further comprises formulating (or administering) a JAK inhibitor or IL-6 signaling inhibitor for the patient.

  In some embodiments, the efficacy is an improvement in patient survival.

  In some embodiments, the efficacy is an improvement in patient progression-free survival.

  As used herein, progression-free survival refers to the total time during and after treatment of a solid tumor where the patient lives with the disease but does not worsen.

In some embodiments, this application:
(A) selecting a solid tumor patient having a serum concentration of C-reactive protein (CRP) that is about 10 μg / mL or greater;
(B) providing a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of an inhibitor of ruxolitinib or a pharmaceutically acceptable salt thereof, wherein the treatment is survival or progression free Providing patients with extended survival.

In addition, this application:
(A) selecting patients with solid tumors with an improved Glasgow prognostic score of 1 or 2;
(B) also provides a method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of an inhibitor of ruxolitinib or a pharmaceutically acceptable salt thereof, wherein the treatment is survival or progression free Providing patients with extended survival.

  In some embodiments, the solid tumor referred to in each of the methods includes prostate cancer, renal cancer, liver cancer, colon cancer, rectal cancer, renal cancer, colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer ( For example, metastatic lung cancer, mesothelioma, or non-small cell lung cancer (NSCLC)), head and neck cancer, thyroid cancer, glioblastoma, Kaposi's sarcoma, Castleman lymphoma, melanoma, esophageal cancer, gastroesophageal cancer, Cervical cancer, hepatocellular carcinoma, endometrial cancer, urothelial cancer (eg, urinary bladder cancer and renal pelvis cancer, eg, transitional cell carcinoma (TCC), or ovarian cancer).

  In some embodiments, solid tumors further include solid tumors characterized by expression of mutant JAK2 such that it has at least one mutation in the pseudokinase domain (eg, JAK2V617F).

  In some embodiments, the solid tumor is pancreatic cancer, prostate cancer, colon cancer, gastric cancer or lung cancer.

  In some embodiments, the solid tumor is pancreatic cancer.

  In some embodiments, the solid tumor is a relapsed or refractory pancreatic adenocarcinoma.

  In some embodiments, the solid tumor is metastatic pancreatic adenocarcinoma.

  In some embodiments, the solid tumor is advanced pancreatic adenocarcinoma.

  In some embodiments, the solid tumor is a relapsed or refractory metastatic pancreatic adenocarcinoma.

  In some embodiments, the solid tumor is a relapsed or refractory advanced pancreatic adenocarcinoma.

  In some embodiments, the solid tumor is prostate cancer.

  In some embodiments, the solid tumor is colon cancer.

  In some embodiments, the solid tumor is gastric cancer.

  In some embodiments, the solid tumor is lung cancer.

  In some embodiments, the solid tumor is endometrial cancer.

  In some embodiments, the solid tumor is non-small cell lung cancer.

  In another aspect, the application provides a method of prolonging survival or progression-free survival in a patient having diffuse large B-cell lymphoma, wherein the patient is of C-reactive protein (CRP). Having elevated serum concentration, the method comprising administering to the patient a Janus kinase (JAK) inhibitor or an IL-6 signaling inhibitor, wherein the administration is dependent on the patient's survival or absence. Prolong survival for exacerbations.

  The application also provides a method of prolonging survival or progression free survival in patients with diffuse large B-cell lymphoma, wherein the patient has an improved Glasgow Prognostic Score (mGPS) of 1 or 2. And the method comprises administering to the patient a JAK inhibitor or an IL-6 signaling inhibitor, wherein the administration prolongs the patient's survival or progression-free survival.

  The application further provides a method of treating diffuse large B cell lymphoma in a patient in need of treatment, wherein the patient has an improved Glasgow Prognostic Score (mGPS) of 1 or 2. The method comprises administering a Janus kinase (JAK) inhibitor or an IL-6 signaling inhibitor to the patient.

In addition, this application:
(A) selecting a lymphoma patient having a serum concentration of C-reactive protein (CRP) that is greater than or equal to the median CRP baseline serum concentration for a patient population having a solid tumor;
(B) providing a method of treating diffuse large B-cell lymphoma comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

In addition, this application:
(A) selecting a lymphoma patient having a serum concentration of C-reactive protein (CRP) that is about 10 μg / mL or greater;
(B) There is also provided a method of treating diffuse large B-cell lymphoma comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or IL-6 signaling inhibitor.

In addition, this application:
(A) selecting patients with lymphoma with an improved Glasgow prognostic score of 1 or 2;
(B) providing a method of treating diffuse large B-cell lymphoma comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.

  The application also provides a method for predicting the efficacy of a treatment using a JAK inhibitor or an IL-6 signaling inhibitor for a patient with diffuse large B-cell lymphoma, the method comprising: Comparing the serum concentration of C-reactive protein (CRP) of the patient to the baseline serum concentration of CRP in a patient population with lymphoma, wherein the serum CRP concentration of the patient above the baseline serum concentration is The efficacy of treatment using JAK inhibitors or IL-6 signaling inhibitors for patients is shown.

  In some embodiments, the diffuse large B cell lymphoma is activated B cell-like (ABC) diffuse large B cell lymphoma (ABC-DLBCL). In some embodiments, the diffuse large B cell lymphoma is germinal center B cell (GCB) diffuse large B cell lymphoma (GCB-DLBCL).

  In some embodiments, any of the methods can include administering one or more additional chemotherapeutic agents.

  In some embodiments, the one or more chemotherapeutic agents are selected from antimetabolites, topoisomerase 1 inhibitors, platinum analogs, taxanes, anthracyclines, EGFR inhibitors, and combinations thereof.

  In some embodiments, antimetabolite agents include capecitabine, gemcitabine, and fluorouracil (5-FU).

  In some embodiments, taxanes include paclitaxel, Abraxane® (paclitaxel-protein binding particles for injectable suspensions), and Taxotere® (docetaxel).

  In some embodiments, platinum analogs include oxaliplatin, cisplatin, and carboplatin.

  In some embodiments, topoisomerase 1 inhibitors include irinotecan and topotecan.

  In some embodiments, anthracyclines include doxorubicin or a liposomal formulation of doxorubicin.

  In some embodiments, the one or more chemotherapeutic agents are selected from one or more additional chemotherapeutic agents, and capecitabine, gemcitabine, Abraxane® (paclitaxel-protein for injectable suspensions) Binding particles), docetaxel, fluorouracil (5-FU), oxaliplatin, cisplatin, carboplatin, irinotecan, topotecan, paclitaxel, leucovorin, doxorubicin, and combinations thereof. In some embodiments, the chemotherapeutic agent is FOLFIRINOX (5-FU, leucovorin, irinotecan and oxaliplatin).

  In some embodiments, the chemotherapeutic agent is FOLFOX (folinic acid (leucovorin), 5-FU, and oxaliplatin (Eloxatin).

  In some embodiments, the one or more additional chemotherapeutic agent is capecitabine.

  In some embodiments, the one or more additional chemotherapeutic agents are capecitabine and oxaloplatin.

  In some embodiments, the one or more additional chemotherapeutic agents is fluorouracil (5-FU).

  In some embodiments, the one or more additional chemotherapeutic agents are gemcitabine and Abraxane® (paclitaxel-protein coupled particles for injectable suspensions).

  Examples of the JAK inhibitor or IL-6 signaling inhibitor include pharmaceutically acceptable salts of the inhibitor. As used herein, a “pharmaceutically acceptable salt” refers to a derivative of a compound, whose parent compound converts an existing acid or base moiety into its salt form. Is modified by Examples of pharmaceutically acceptable salts include inorganic acid salts or organic acid salts of basic residues such as amines; alkali salts or organic salts of acidic residues such as carboxylic acids. It is not limited to. The pharmaceutically acceptable salts of the present invention include the non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. In general, such salts react the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two. In general, non-aqueous media such as ether, ethyl acetate, alcohols (eg, methanol, ethanol, isopropanol, or butanol) or acetonitrile (ACN) are preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17th ed. , Mack Publishing Company, Easton, Pa. 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

  As used herein, the terms “individual” or “patient” used interchangeably refer to any animal, such as a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig. , Cow, sheep, horse, or primate, most preferably a human.

As used herein, the phrase “therapeutically effective amount” refers to a biological response or drug in a tissue, system, animal, individual or human being sought by a researcher, veterinarian, physician or other clinician. Refers to the amount of active compound or pharmaceutical agent that elicits a response, which response includes one or more of the following:
(1) prevention of disease; for example, prevention of a disease, condition or disorder in an individual who may promote the onset of the disease, condition or disorder but has not yet experienced or shown the pathology or symptom of the disease;
(2) Inhibiting disease; for example, inhibiting disease, condition or disorder in an individual experiencing or showing pathology or symptom of disease, condition or disorder (ie, further pathology and / or symptom symptom) And (3) ameliorating the disease; for example, ameliorating the disease, condition or disorder in an individual experiencing or presenting the pathology or symptom of the disease, condition or disorder (ie, Reversing pathology and / or general symptoms).

Pharmaceutical formulations and dosage forms When used as a medicament, the JAK inhibitor or IL-6 signaling inhibitor can be administered in the form of a pharmaceutical composition. These compositions can be prepared in a manner known in the pharmaceutical industry and are administered by various routes depending on whether local or systemic treatment is required and on the area to be treated. be able to. Administration can be local (eg transdermal, epithelial, ocular and transmucosal, eg intranasal, vaginal and rectal delivery), lung (eg by inhalation of powder or aerosol or insufflation, eg by nebulizer; intratracheal or nasal) Internal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion; or intracranial, eg, intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose or it can be, for example, by a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated sacks and gloves may also be useful.

  The method also comprises a pharmaceutical composition comprising one or more JAK inhibitors or IL-6 signaling inhibitors as active ingredients in combination with one or more pharmaceutically acceptable carriers (excipients). You can also use things. In making the compositions of the invention, the active ingredient is typically mixed with or diluted with an excipient, or such as in a capsule, sachet, paper, or other container. Encapsulated in the form of the carrier. Where the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, and serves as a vehicle, carrier or medium for the active ingredient. Thus, the composition comprises tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as solids or in liquid media), For example, it can be in the form of ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders containing up to 10% by weight of the active compound.

  In preparing a formulation, the active compound can be milled to a suitable particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be ground to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted, for example to about 40 mesh, by milling to provide a substantially uniform dispersion in the formulation.

  Some examples of suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose , Water, syrup, and methylcellulose. The formulation further includes: lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers and suspending agents; preservatives such as methyl- and propylhydroxy-benzoates; sweeteners; and flavoring agents. can do. The compositions of the present invention can be formulated so that the active ingredient is rapidly released, sustained released, or delayed released after administration to a patient by using procedures known in the art.

  The composition can be formulated in unit dosage form, with each dose ranging from about 5 to about 1000 mg (1 g), more usually from about 100 to about 500 mg, about 10 mg, about 15 mg, about 20 mg, or about Contains 25 mg of active ingredient. The term “unit dosage form” refers to physically discrete units suitable as unit dosages for human subjects and other mammals, each unit combined with a suitable pharmaceutical excipient as desired. A predetermined amount of active substance calculated to produce a therapeutic effect of

  The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. However, the amount of compound actually administered is usually determined by the physician in the relevant context, such as the condition to be treated, the route of administration selected, the actual compound administered, the age, weight and response of each patient, It will be understood that this is determined according to the severity of the patient's symptoms.

  To produce a solid composition such as a tablet, the main active ingredient is mixed with pharmaceutical excipients to produce a solid preformulation composition containing a homogeneous mixture of the compounds of the invention. When the pre-formulated composition is homogeneous, the active ingredient is typically formulated so that the composition can be easily divided into unit dosage forms such as tablets, pills and capsules with comparable effects. Disperse uniformly throughout the object. This solid preformulation composition is then divided into unit dosage forms of the type described above containing, for example, from about 0.1 to about 1000 mg of active ingredient.

  Tablets or pills can be coated or otherwise formulated to provide a dosage form that provides the benefit of prolonged action. For example, a tablet or pill can comprise an inner dosage component and an outer dosage component (the latter being in the form of an envelope over the former). The two dosage components can be separated by an enteric layer that helps to resist disintegration in the stomach and to pass through the duodenum or delay release without compromising the internal components. Various materials can be used for such enteric layers and coatings, such as several polymeric acids, and polymeric acids such as shellac, cetyl alcohol and cellulose acetate. Mixtures with materials are mentioned.

  Liquid forms in which compounds and compositions can be incorporated for oral or infusion administration include aqueous solutions, preferably flavored syrups, aqueous or oily suspensions, and edible oils such as cottonseed oil, sesame oil, Examples include flavored emulsions with coconut oil or peanut oil, and elixirs and similar pharmaceutical vehicles.

  Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid composition may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the composition is administered for local or systemic effect by the oral or nasal respiratory route. The composition can be nebulized by use of an inert gas. The solution to be nebulized may be inhaled directly from the nebulizer, or the nebulizer may be attached to a facial mask tent or intermittent positive pressure breathing device. Solution, suspension, or powder compositions can be administered orally or nasally from devices that deliver the formulation in an appropriate manner.

  The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of administration, eg, prophylaxis or treatment, the condition of the patient, the method of administration and the like. For therapeutic use, the composition can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. Effective doses will vary at the discretion of the attending physician depending on the condition of the disease being treated and factors such as, for example, the severity of the disease, the age, weight and general condition of the patient.

  The composition administered to the patient can be in the form of the pharmaceutical composition described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being mixed with a sterile aqueous carrier prior to administration. The pH of the compound preparation is typically 3-11, more preferably 5-9, and most preferably 7-8. It will be appreciated that the use of certain of the above excipients, carriers, or stabilizers forms a pharmaceutical salt.

  The therapeutic dosage of the compounds of the invention can vary according to, for example, the particular application for which the treatment is being performed, the method of administering the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of the compound of the invention in the pharmaceutical composition may vary depending on a number of factors, such as dosage, chemical nature (eg, hydrophobicity), and route of administration. For example, the compounds of the present invention can be provided in an aqueous physiological buffer solution containing from about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dose ranges are from about 1 μg / kg body weight / day to about 1 g / kg body weight / day. In some embodiments, the dose range is from about 0.01 mg / kg body weight / day to about 100 mg / kg body weight / day. The dosage will probably depend on the type and extent of the disease or disorder, the overall health status of the particular patient, the relative biological effectiveness of the selected compound, the formulation of the excipient, and its route of administration. It may depend on such variable conditions. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  The compositions of the present invention can further include one or more additional pharmaceutical agents such as chemotherapeutic agents, steroids, anti-inflammatory compounds, or immunosuppressive agents, examples of which are as described above.

Combination Therapy One or more additional pharmaceutical agents such as chemotherapeutic agents, anti-inflammatory agents, steroids, immunosuppressive agents, and Bcr-Abl, Flt-3, RAF and FAK kinase inhibitors, such as those herein incorporated by reference in their entirety. The ones described in WO 2006/056399, or other agents incorporated into the US, can be used in combination with the dosage forms described herein for use in the methods described herein. The one or more additional pharmaceutical agents can be administered to the patient simultaneously or sequentially.

  Examples of chemotherapeutic agents include proteosome inhibitors (eg bortezomib), thalidomide, lenaliimide, and DNA-damaging agents such as melphalan, doxorubicin, cyclophosphamide, vincristine, etoposide, carmustine and the like.

  Examples of steroids include corticosteroids such as dexamethasone or prednisone.

  In some embodiments, one or more dosage forms can be used in combination with one or more non-steroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the NSAID is aspirin, diflunisal, salsalate, ibuprofen, naproxen, fenoprofen, ketoprofen, oxaprozin, indomethacin, tolmetine, sulindac, etodolac, ketodolac, piroxicam, meloxicam Selected from acetaminophen, celecoxib, and combinations thereof.

  Examples of Bcr-Abl inhibitors include genus and species compounds disclosed in US Pat. No. 5,521,184, WO 04/005281, and US patent application 60 / 578,491, and pharmaceutically Acceptable salts are mentioned, the entirety of which is incorporated herein by reference.

  Examples of suitable Flt-3 inhibitors include the compounds disclosed in WO03 / 037347, WO03 / 099771, and WO04 / 046120 and their pharmaceutically acceptable salts, the entirety of which are by reference. Incorporated herein.

  Examples of suitable RAF inhibitors include the compounds disclosed in WO 00/09495 and WO 05/028444 and their pharmaceutically acceptable salts, both of which are incorporated herein by reference in their entirety. .

  Examples of suitable FAK inhibitors include the compounds disclosed in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402 and pharmaceutically acceptable salts thereof. Which are incorporated herein by reference in their entirety.

  In some embodiments, one or more of the dosage forms of the invention are used in combination with other kinase inhibitors, such as imatinib, particularly to treat patients resistant to imatinib or other kinase inhibitors. be able to.

  In some embodiments, one or more dosage forms of the invention can be used in combination with a chemotherapeutic agent in the treatment of solid tumors, with a response to the chemotherapeutic agent alone without exacerbating its toxic effects. In comparison, the therapeutic response can be improved. Examples of additional pharmaceutical agents include, but are not limited to, melphalan, melphalan + prednisone [MP], doxorubicin, dexamethasone, and Velcade (bortezomib). Additional additional agents used in therapy include Bcr-Abl, Flt-3, RAF, mTor, EGFR, PI3K-delta, and FAK kinase inhibitors. An additive or synergistic effect is a desirable result of the combination of a dosage form of the invention and an additional agent. The agents can be combined with a JAK inhibitor or an IL-6 signaling inhibitor in a single or continuous dosage form, or the agents can be administered simultaneously or sequentially as separate dosage forms.

  In some embodiments, a corticosteroid, such as dexamethasone, is administered to a patient in combination in a dosage form of the invention, where dexamethasone is administered intermittently rather than continuously.

  In some further embodiments, the combination of one or more JAK inhibitors or IL-6 signaling inhibitors and other therapeutic agents is administered to the patient before, during, and / or after bone marrow transplant or stem cell transplant. Can be administered.

  In some embodiments, the additional therapeutic agent is fluocinolone acetonide (Retisert®) or rimexolone (AL-2178, Vexol, Alcon).

  In some embodiments, the additional therapeutic agent is cyclosporine (Restasis®).

  In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the corticosteroid is triamcinolone, dexamethasone, fluocinolone, cortisone, prednisolone, or flumethrone.

  In some embodiments, the additional therapeutic agent is Dehydrex ™ (Holls Labs), Civamide (Opko), Sodium Hyaluronate (Vismed, Lantiobio / TRB Chemedia), Cyclosporine (ST-603, Sirion Therapeutic G). (T) (testosterone, Argentis), AGR1012 (P) (Argentis), ecabet sodium (Senju-Ista), gefarnate (Santen), 15- (s) -hydroxyeicosatetraenoic acid (15 (S) -HETE ), Sevillemin, doxycycline (ALTY-0501, Alacrity), minocycline, iDestrin ™ (NP503) 1, Nascent Pharmaceuticals), Cyclosporin A (Nova22007, Novagali), Oxytetracycline (Duramycin, MOLI1901, Lantbio), CF101 (2S, 3S, 4R, 5R) -3,4-dihydroxy-5- [6-[(3 -Iodophenyl) methylamino] purin-9-yl] -N-methyl-oxolane-2-carbamyl, Can-Fite (Biopharma), voclosporin (LX212 or LX214, Lux Biosciences), ARG103 (Agents), RX-10045 ( Synthetic resolvin analogues, Resolvyx), DYN15 (Dynamis Therapeutics), Riboglitazone (DE011, Daiic) i Sanko), TB4 (RegeneRx), OPH-01 (Ophthalmis Monaco), PCS101 (Pericor Science), REV1-31 (Evolutec), Lacritin (Senju), Rebamipide (Otsuka-OITari, Otsuka-OTAT), -2 (University of Pennsylvania and Temple University), pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednol etabonate, rituximab, dicafozole tetrasodium (INS365, Inspire), KLS-0611 (Kissei Pharmaceuticals) Androsterone, Anakinra, Efalizumab, Mycophenolate Natri Arm, etanercept (Embrel (registered trademark)), hydroxychloroquine, NGX267 (TorreyPines Therapeutics), Actemra, gemcitabine, oxaliplatin, is selected from L- asparaginase or thalidomide.

  In some embodiments, the additional therapeutic agent is an anti-angiogenic agent, a cholinergic agonist, a TRP-1 receptor modulator, a calcium channel blocker, a mucin secretagogue, a MUC1 stimulator, a calcineurin inhibitor, cortico Steroid, P2Y2 receptor agonist, muscarinic receptor agonist, mTOR inhibitor, another JAK inhibitor, Bcr-Abl kinase inhibitor, Flt-3 kinase inhibitor, RAF kinase inhibitor, and FAK kinase inhibitor, For example, those described in WO 2006/056399, which is incorporated herein by reference in its entirety. In some embodiments, the additional therapeutic agent is a tetracycline derivative (eg, minocycline or doxyline). In some embodiments, the additional therapeutic agent binds FKBP12.

  In some embodiments, the additional therapeutic agent is an alkylating agent or DNA cross-linking agent; an antimetabolite / demethylating agent (eg, 5-fluorouracil, capecitabine or azacitidine); an anti-hormonal therapeutic agent (eg, a hormone) Receptor antagonists, SERM or aromatase inhibitors); mitotic inhibitors (eg vincristine or paclitaxel); topoisomerase (I or II) inhibitors (eg mitoxantrone and irinotecan); apoptosis-inducing factors (eg ABT-737); Nucleic acid therapeutic agents (eg antisense or RNAi); nuclear receptor ligands (eg agonists and / or antagonists: all-trans retinoic acid or bexarotene); epigenetic targeting agents such as histone deacetylase inhibitors Agents (e.g. vorinostat) hypomethylation agent (e.g. decitabine); or EGFR inhibitor (erlotinib); protein stability modifiers, for example Hsp90 inhibitors, ubiquitin and / or ubiquitin-like conjugated or uncoupling molecules.

  In some embodiments, additional therapeutic agents include antibiotics, antiviral agents, antifungal agents, anesthetic agents, anti-inflammatory agents such as steroidal and non-steroidal anti-inflammatory agents, antiallergic agents. Examples of suitable agents include aminoglycosides such as amikacin, gentamicin, tobramycin, streptomycin, netilmicin, and kanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin, and Enoxacin; naphthyridine; sulfonamide; polymyxin; chloramphenicol; neomycin; paramomycin; colistimate; bacitracin; vancomycin; tetracycline; rifampin and its derivatives (“rifampin”); cycloserine; β-lactam; cephalosporin; Flucytosine; natamycin; miconazole; ketoconazole; corticosteroid; Enaku; flurbiprofen; ketorolac; suprofen; cromolyn; lodoxamide; levocabastine; naphazoline; antazoline; pheniramine; or azalide antibiotic and the like.

  Certain features of the invention that are described in the context of separate embodiments for purposes of clarity have the meaning of a single implementation (as if the embodiments in the specification were recited as multiple dependent claims). It is further appreciated that combinations of forms may also be provided.

Example Example 1
Life-prolonging effects in pancreatic patients with C-reactive protein (CRP) levels above the median baseline This study examines safety designed to confirm the safety of capecitabine / luxolitinib combination for this patient population It consisted of a blinded trial (consisting of 1 to 2 cohorts) followed by a randomized double-blind trial with 2 treatment groups. All subjects received capecitabine therapy in addition to study drug. In the safety induction phase, the study drug was open-label luxolitinib phosphate; for randomized trials, the blinded study drug was ruxolitinib phosphate or its placebo. It was.

  The subject's treatment consisted of repeating a 21 day cycle. Capecitabine was self-administered during the first 14 days of each cycle, and the test drug was self-administered during all cycles. The treatment cycle continued as long as the regimen was tolerated and subjects did not meet the discontinuation criteria. In the case of disease hate, capecitabine therapy was discontinued; subjects continued to receive study drug. Subjects who discontinued treatment with study drug were followed for subsequent treatment regimens and survival.

Test design The parameters of the tests performed by those skilled in the art are described below.

Safety run-in:
Cohort 1: 9 subjects are enrolled to receive capecitabine 2000 mg / m ² / day (as 1000 mg / m ² BID) + luxolitinib phosphate 15 mg BID on a free base basis. If more than two subjects in Cohort 1 experience DLT within the first cycle of treatment (21 days), the second cohort is enrolled.

Cohort 2: 9 additional subjects will receive capecitabine 2000 mg / m ² / day (as 1000 mg / m ² BID) + luxolitinib phosphate 10 mg BID on a free base basis.

  If the toxicity that occurs is clearly related to capecitabine, a lower dose of capecitabine is considered than or in addition to the lower dose of ruxolitinib.

  Therefore, the dose selected for the randomized portion of the study is a tolerable dose that does not require at least two-thirds of subjects tested at that dose to reduce the dose within 21 days. is there. If more than 3 DLTs occur in Cohort 1 and Cohort 2, the randomized portion of the study will not be enrolled.

Randomized part of the study:
120 subjects were randomized 1: 1 into two treatment groups:
Group 1: capecitabine + study drug (luxolitinib phosphate)
Group 2: capecitabine + study drug (placebo)
Subjects, researchers, and responsible organizations are blinded to treatment assignment.
The starting dose of study drug is the dose selected during the safety induction phase.

Combination therapy, dosage and administration method:
Capecitabine (as an open label commercial product) is self-administered orally twice daily (BID) for the first 14 days of each cycle. Study drug (luxolitinib or placebo) is self-administered orally twice daily (BID) during a total 21-day cycle. The study drug being introduced to safety is used in the randomized part of the study, and in each subject, the dose of study drug or capecitabine is reduced during the course of treatment based on a laboratory assessment of safety. May be. Subjects with stable safety parameters can individually meet the conditions for increasing the dose of the test agent according to a defined algorithm.

  Participation period: Participation of study subjects is expected to average 4 to 8 months.

  Study population: Subjects with recurrent or refractory metastatic pancreatic adenocarcinoma.

Selection criteria:
• Diagnosis of metastatic pancreatic cancer; subject must have histologically confirmed measurable or evaluable disease • Over 60 Karnovsky performance status • Subject has metastatic pancreatic cancer First-line drug for gemcitabine treatment must have failed:
Alternative chemotherapeutic agents are an acceptable alternative as first-line therapy in events where the subject is not tolerated or ineligible for gemcitabine administration.> 2 weeks or more after completion of past chemotherapy Elapsed and subject must have recovered or be in a new stable baseline state from any associated toxicity

Exclusion Criteria • Two or more previous chemotherapy regimens for metastatic disease (excluding adjuvant therapy)
• Evidence of CNS metastases (if not stable for> 3 months) or history of uncontrollable seizures • Ongoing or past radiotherapy given as second-line treatment • Basal cell carcinoma of the skin or Other active malignancies other than squamous cell carcinoma • Any condition of the upper gastrointestinal tract that prevents food swallowing or prevents oral medications • A recent history of intestinal obstruction (≦ 3 months) • Past severe treatment for fluoropyrimidine Response, known DPD deficiency, or sensitivity to other known 5-FUs • Inadequate kidney, liver and bone marrow function:
・ ANC <1500 / mm ³
Platelet <75,000 / mm ³
AST / ALT> 2.5 X ULN; or if liver metastases are observed> 5 X ULN
・ Total bilirubin> 1.5 X ULN
・ Creatinine clearance <50cc / min

Target number of plans:
About 9-18 subjects in the safety introductory part of the study, followed by 1: 1 in each of the two treatment groups of 120 subjects in the randomized part of the study.

Test schedule / procedure:
On the first day of each cycle, a laboratory visit is conducted to include physical examination and clinical examination. In addition, a study visit is performed weekly during cycle 1 and cycle 2 and once in the middle of all subsequent cycles (about day 10). Reassessment of tumor size (typically by computed tomography) is performed every 6 weeks during study participation. Medical effect assessments by patients are collected at several laboratory visits. The first day of each test cycle is the start of the 14-day course of capecitabine. If capecitabine is discontinued, the test cycle continues to follow the 21 day repeat schedule. After discontinuation of all study treatments, the assessment is discontinued and followed for survival and subsequent anticancer therapy.
Planned number of test facilities: approx. 50
Test duration assessed: 20 months Statistical method: Survival data are analyzed by Kaplan-Meier method after 95 events. The hazard ratio and its 95% confidence interval are determined based on the log rank test and its variance. If the true hazard ratio is 0.6, the power to detect the difference in survival time between groups 1 and 2 is 88% at a sample size of 60 subjects per group. This assumes a one-sided alpha of 0.1, an expected survival of 4 months in the subject group, an 8-month enrollment, and an 8-month follow-up from the last subject. When half the target number of deaths is achieved, a pre-planned interim analysis is performed for futileness.

Randomized trial results Baseline C-reactive protein (CRP) levels were measured for each patient in groups 1 and 2 prior to treatment. Serum CRP concentration was measured by RMB (RBM multiplexed Luminex®) commercial assay (Myriad RBM). The subsidiary is Myriad RBM. There are a number of known commercial clinical assays for measuring CRP. The Myriad RBM CRP assay was found to correlate with the Luminex CRP assay using a commercially available reagent (Millipore) and a clinical Quest Diagnostics CRP assay. Baseline CRP levels were calculated for each patient. Patients included two groups. Group 1 included all patients randomized and administered the study drug. Group 2 is a small subset of patients who passed the screening and were randomized but did not receive the study drug. In Group 1, the CRP level was the last tested CRP level taken before the first dose of study drug. For Group 2, the last available value of CRP was taken from, for example, a screening procedure, if available. Median values were calculated using normal statistical methods known to those skilled in the art, along with baseline CRP levels for all patients. The median baseline CRP for the patient population was 13 μg / mL.

  Survival data were analyzed statistically as described by Kaplan-Meier analysis of overall survival using Cox proportional hazards model score test. Table 1 and FIG. 1 show the results for patients whose baseline CRP was 13 μg / mL or less, and Tables 2 and 2 show the results for patients whose baseline CRP exceeded 13 μg / mL. Subjects who were censored were patients who were unable to follow up or had no deaths recorded prior to the deadline of clinical data.

［１］ハザード比及び９５％ＣＩは、同順位（ｔｉｅ）のために使用されるＥｆｒｏｎ法と共にＣｏｘ回帰モデルを使用して推定した。
［２］時間の中央値及び９５％ＣＩは、Ｂｒｏｏｋｍｅｙｅｒ及びＣｒｏｗｌｅｙを使用して推定した。
［３］片側ｐ値は、Ｃｏｘ比例ハザードモデルのスコア検定に基づいて計算した。

［１］ハザード比及び９５％ＣＩは、同順位（ｔｉｅ）のために使用されるＥｆｒｏｎ法と共にＣｏｘ回帰モデルを使用して推定した。
［２］時間の中央値及び９５％ＣＩは、Ｂｒｏｏｋｍｅｙｅｒ及びＣｒｏｗｌｅｙを使用して推定した。
［３］片側ｐ値は、Ｃｏｘ比例ハザードモデルのスコア検定に基づいて計算した。

［１］無増悪生存期間は、ＲＥＣＩＳＴ １．１によって、死亡または進行性疾患の初回発生と定義した。
［２］ハザード比及び９５％ＣＩは、同順位（ｔｉｅ）のために使用されるＥｆｒｏｎ法と共にＣｏｘ回帰モデルを使用して推定した。
［３］時間の中央値及び９５％ＣＩは、Ｂｒｏｏｋｍｅｙｅｒ及びＣｒｏｗｌｅｙを使用して推定した。
［４］両側ｐ値は、Ｃｏｘ比例ハザードモデルのスコア検定に基づいて計算した。

［１］無増悪生存期間は、ＲＥＣＩＳＴ １．１によって、死亡または進行性疾患の初回発生と定義した。
［２］ハザード比及び９５％ＣＩは、同順位（ｔｉｅ）のために使用されるＥｆｒｏｎ法と共にＣｏｘ回帰モデルを使用して推定した。
［３］時間の中央値及び９５％ＣＩは、Ｂｒｏｏｋｍｅｙｅｒ及びＣｒｏｗｌｅｙを使用して推定した。
［４］両側ｐ値は、Ｃｏｘ比例ハザードモデルのスコア検定に基づいて計算した。 In addition, progression free survival was similarly analyzed by Kaplan-Meier analysis of progression free survival using the Cox proportional hazards model score test. Table 3 and FIG. 3 show the results for patients whose baseline CRP was 13 μg / mL or less, and Tables 4 and 4 show the results for patients whose baseline CRP exceeded 13 μg / mL.

[1] Hazard ratio and 95% CI were estimated using a Cox regression model with the Efron method used for tie.
[2] Median time and 95% CI were estimated using Brookmeyer and Crowley.
[3] The one-sided p-value was calculated based on the Cox proportional hazard model score test.

[1] Hazard ratio and 95% CI were estimated using a Cox regression model with the Efron method used for tie.
[2] Median time and 95% CI were estimated using Brookmeyer and Crowley.
[3] The one-sided p-value was calculated based on the Cox proportional hazard model score test.

[1] Progression-free survival was defined by RECIST 1.1 as the first occurrence of death or progressive disease.
[2] Hazard ratio and 95% CI were estimated using the Cox regression model with the Efron method used for tie.
[3] Median time and 95% CI were estimated using Brookmeyer and Crowley.
[4] The two-sided p-value was calculated based on the Cox proportional hazard model score test.

[1] Progression-free survival was defined by RECIST 1.1 as the first occurrence of death or progressive disease.
[2] Hazard ratio and 95% CI were estimated using the Cox regression model with the Efron method used for tie.
[3] Median time and 95% CI were estimated using Brookmeyer and Crowley.
[4] The two-sided p-value was calculated based on the Cox proportional hazard model score test.

１）ハザード比及び９５％ＣＩは、同順位（ｔｉｅ）のために使用されるＥｆｒｏｎ法と共にＣｏｘ回帰モデルを使用して推定した。
２）両側ｐ値はＣｏｘ比例ハザードモデルのスコア検定に基づいて計算した。 Table 5 shows the results of Cox regression analysis within the CRP> 13 mg / L subgroup. The regression model fits the data well (p = 0.022) and explains the baseline characteristics in the model, and the observed HR in favor of ruxolitinib is mostly maintained (HR 0.50, 95 % CI: 0.26-0.96; p = 0.037).

1) Hazard ratio and 95% CI were estimated using Cox regression model with Efron method used for tie.
2) The two-sided p-value was calculated based on the Cox proportional hazard model score test.

１）同順位を処理するＥｆｒｏｎ法を使用するスコア試験に基づいている。
２）検定する３つの仮説を想定している。 Table 6 shows the results of a Cox regression analysis that encompasses all of the above subgroups, but for the three pre-designated subgroups based on the hypothesis that ruxolitinib provides disproportionate effectiveness, An action test was also performed. In these three subgroups, only CRP (> 13 mg / L) above the median test population appears as a defining factor with a bilateral Bonferroni corrected p-value of 0.032.

1) Based on a score test using the Efron method to process ties.
2) Assume three hypotheses to test.

Progression-free survival In a treatment intention analysis involving all randomized patients, the HR for progression-free survival (PFS) was 0.75 (CI: 0.52, 1.1, p = 0.14). It was. Evaluation of PFS in patients with CRP> 13 mg / L showed an HR of 0.62 (95% CI: 0.35-1.1, p = 0.10). The probability of progression-free survival at 3, 6 and 9 months was 35%, 21% and 11% in the ruxolitinib group, 13%, 5% and 0% in the placebo group. Assessment of PFS in patients with CRP <13 mg / L showed a hazard ratio of 0.82 (95% CI: 0.47-1.41, p = 0.47). The probability of PFS at 3, 6 and 9 months was 39%, 25% and 10% in the ruxolitinib group and 38%, 14% and 4% in the placebo group.

Improved Glasgow Prognostic Score (mGPS)
The pre-specified subgroup analysis used the median CRP (13 mg / L) of the entire study population as a cut-off; in the subsequent analysis, a 10 mg / L cut-off consistent with mGPS , Using generally accepted criteria for clinically meaningful elevation (McMillan et al 2007; FDA Guidance on CRP Assays). For patients with CRP> 10 mg / L (N = 70), the favorable HR for ruxolitinib is 0.60 (95% CI: 0.351-1.028, bilateral p = 0.06) and CRP ≦ 10 mg In patients with / L (N = 51), the HR was 0.91 (95% CI: 0.46-1.74, p = 0.77).

In Kaplan-Meier analysis of OS based on mGPS, there was a meaningful separation between ruxolitinib and placebo group in OS by increasing mGPS. In patients with 0 mGPS (N = 51), the HR was 0.91 (95% CI: 0.46-1.74, p = 0.77). In patients with 1 mGPS (N = 34), the HR was 0.71 (95% CI: 0.32-1.54, p = 0.39). In patients with 2 mGPS (N = 36), the HR was 0.49 (95% CI: 0.23-1.07, p = 0.06). Kaplan-Meier curves for all three groups are shown in FIG.

Objective response rate Water of systemic tumor tissue volume change as assessed by the treatment efficacy criteria for solid tumors (RECIST v1.1, one-dimensional index of target lesions) for patients with at least one post-baseline tumor assessment From the fall plot, it was found that patients treated with ruxolitinib were likely to show tumor shrinkage or stabilization as the best response to therapy. Placebo-treated patients with CRP> 13 mg / L are long enough to receive a post-baseline scan, most do not survive (N = 11), and only a few patients respond or have stabilized disease (36.4%) compared to the majority of patients with CRP> 13 mg / L who received ruxolitinib received a post-baseline assessment (N = 19), the majority of which had disease stabilization (68.4%).

工程１．ｔｅｒｔ−ブチル（４Ｓ）−２，２−ジメチル−４−ビニル−１，３−オキサゾリジン−３−カルボキシレート
テトラヒドロフラン（１４０ｍＬ）中メチルトリフェニルホスホニウムブロミド（５．６３ｇ、１５．８ｍｍｏｌ）の懸濁液に対して、ヘキサン（７．３５ｍＬ、１８．４ｍｍｏｌ）中２．５Ｍのｎ−ブチルリチウムを加えた。その濃い赤色溶液を０℃で１時間撹拌した。次いで、テトラヒドロフラン（７．３ｍＬ）中ｔｅｒｔ−ブチル（４Ｒ）−４−ホルミル−２，２−ジメチル−１，３−オキサゾリジン−３−カルボキシレート（Ａｌｄｒｉｃｈ製、３．０１ｇ、１３．１ｍｍｏｌ）溶液を、０℃で滴加した。その赤色溶液を室温まで温め、そして１２時間撹拌した。ヘキサンを、その反応混合物に４：１（ｖ／ｖ）の比で加えた。その懸濁液をセライトでろ過し、そのろ液を濃縮した。得られた残留物を、フラッシュクロマトグラフィー（ヘキサン中１０％酢酸エチルで溶出）で精製して、無色の油状物として化合物（１．９２ｇ、６４％）を得た。 Example J1. ((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridin-1-yl} tetrahydro-2H -Pyran-2-yl) acetonitrile

Step 1. tert-Butyl (4S) -2,2-dimethyl-4-vinyl-1,3-oxazolidine-3-carboxylate Suspension of methyltriphenylphosphonium bromide (5.63 g, 15.8 mmol) in tetrahydrofuran (140 mL) To the solution was added 2.5M n-butyllithium in hexane (7.35 mL, 18.4 mmol). The dark red solution was stirred at 0 ° C. for 1 hour. Then, a solution of tert-butyl (4R) -4-formyl-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (manufactured by Aldrich, 3.01 g, 13.1 mmol) in tetrahydrofuran (7.3 mL) was added. At 0 ° C. The red solution was warmed to room temperature and stirred for 12 hours. Hexane was added to the reaction mixture in a ratio of 4: 1 (v / v). The suspension was filtered through celite, and the filtrate was concentrated. The resulting residue was purified by flash chromatography (eluting with 10% ethyl acetate in hexanes) to give the compound (1.92 g, 64%) as a colorless oil.

Step 2. tert-Butyl [(1S) -1- (hydroxymethyl) prop-2-en-1-yl] carbamate tert-Butyl (4S) -2,2-dimethyl-4-vinyl-1,3 in methanol (83 mL) -To the oxazolidine-3-carboxylate (1.90 g, 8.36 mmol) solution, p-toluenesulfonic acid monohydrate (0.80 g, 4.2 mmol) was added at 0 ° C. The mixture was slowly warmed to room temperature overnight. The reaction mixture was diluted with saturated NaHCO ₃ solution, concentrated and then diluted with ethyl acetate. The organic layer is washed with saturated NaHCO ₃ (2 ×) and brine, dried over Na ₂ SO ₄ , filtered and concentrated to give the desired product (1.187 g, 76%) as a colorless oil. Obtained. ¹ H NMR (400 MHz, CDCl ₃ ) δ 5.81 (1H, m), 5.25 (2H, m), 4.90 (1H, m), 4.25 (1H, brs), 3.67 (2H, m), 1.45 (9H, s) ppm.

Step 3. tert-Butyl [(1S) -1-({[1- (hydroxymethyl) prop-2-en-1-yl] oxy} methyl) prop-2-en-1-yl] carbamate [(1S) -1- (hydroxymethyl) prop-2-en-1-yl] carbamate (0.401 g, 2.14 mmol), tris (dibenzylideneacetone) dipalladium (0) (59 mg, 0.064 mmol) , N, N ′-(1S, 2S) -cyclohexane-1,2-diylbis [2- (diphenylphosphino) -1-naphthamide] (150 mg, 0.19 mmol), and 4-dimethylaminopyridine (78 mg, 0 .64 mmol). The reaction mixture was purged with N ₂ three times, then methylene chloride (21.3 mL) and 1.0 M triethylborane in THF (130 μL, 0.13 mmol) were added sequentially. After stirring for 10 minutes, 2-vinyloxirane (0.150 g, 2.14 mmol) was added and the resulting mixture was stirred overnight. The reaction was diluted with dichloromethane and saturated NaHCO ₃ solution. The organic layer was separated, dried over Na ₂ SO ₄ , filtered and concentrated. The crude residue was purified by flash chromatography (eluting with 0-50% ethyl acetate / hexanes) to give the desired product (0.271 g, 49%). ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.85 (1H, m), 5.67 (1H, m), 5.84 to 5.17 (4H, m), 4.83 (1H, m), 4 .30 (1H, br s), 3.83 (1 H, m), 3.69 (1 H, dd, J = 4.5 and 6.9 Hz), 3.54 (2 H, m), 3.36 ( 1H, dd, J = 4.5 and 6.9 Hz), 1.45 (9H, s) ppm.

Step 4. 2-({(2S) -2-[(tert-butoxycarbonyl) amino] but-3-en-1-yl} oxy) but-3-en-1-yl acetate in methylene chloride (10 mL) tert-Butyl [(1S) -1-({[1- (hydroxymethyl) prop-2-en-1-yl] oxy} methyl) prop-2-en-1-yl] carbamate (268 mg, 1.04 mmol ) Was added triethylamine (435 μL, 3.12 mmol). The mixture was cooled to 0 ° C. and acetyl chloride (150 μL, 2.1 mmol) was added dropwise. The reaction was stirred at room temperature for 2 hours and then quenched with water. The organic layer was concentrated and the resulting residue was purified on silica gel (eluting with 20% ethyl acetate / hexanes) to give the desired product (0.26 g, 85%). _{C 10 H 18 NO 3 (M} -100 + H) + for calculated LCMS: m / z = 200.1; Found: 200.1.

Step 5. {(5S) -5-[(tert-Butoxycarbonyl) amino] -5,6-dihydro-2H-pyran-2-yl} methyl acetate Into a 500 mL 2-neck round bottom flask was added benzylidene (dichloro) (1,3 -Dimesitylimidazolidin-2-id-2-yl) (tricyclohexylphosphoranyl) ruthenium (38 mg, 0.044 mmol) was added. After purging 3 times with nitrogen, dichloromethane (anhydrous, 8 mL) was added followed by 2-({(2S) -2-[(tert-butoxycarbonyl) amino] but-3-en-1-yl} oxy) But-3-en-1-yl acetate (265 mg, 0.885 mmol) was added. The reaction mixture was stirred at room temperature for 15 hours. The mixture was concentrated in vacuo. The residue was purified by flash chromatography (eluting with hexane and 25% EtOAc in hexanes) to give the desired product as a brown oil (0.205 g, 85%). _{C 9 H 14 NO 5 (M} + H-Bu + H) + for calculated LCMS: m / z = 216.1; Found: 216.1. ¹ H NMR (300 MHz, CDCl ₃ ) δ 5.94 (0.17H, m), 5.84 (0.83H, m), 5.69 (1H, m), 4.89 (0.13H, m) , 4.70 (0.83H, m), 4.25 (1H, m), 4.05 (4H, m), 3.56 (0.13H, m), 3.38 (0.87H, m ), 2.04 (2.49 H, s), 2.03 (0.51 H, m), 1.38 (9 H, s) ppm (the product is about 5: 1 of the trans- and cis-isomers). Mixture).

Step 6. [(5S) -5-amino-5,6-dihydro-2H-pyran-2-yl] methyl acetate {(5S) -5-[(tert-butoxycarbonyl) amino]-in methylene chloride (5.2 mL) To a solution of 5,6-dihydro-2H-pyran-2-yl} methyl acetate (205 mg, 0.756 mmol), 4.0 M hydrogen chloride in dioxane (1.5 mL, 6.0 mmol) was added. The reaction solution was stirred at room temperature for 6 hours. The solvent was removed under reduced pressure to give the desired product as a white solid. _{C 8 H 14 NO 3 (M} + H) + LCMS calculated for: m / z = 172.1; Found: 172.1.

Step 7. {(5S) -5-[(6-Nitrothieno [3,2-b] pyridin-7-yl) amino] -5,6-dihydro-2H-pyran-2-yl} methyl acetate isopropyl alcohol (1.7 mL ) -7-chloro-6-nitrothieno [3,2-b] pyridine (156 mg, 0.727 mmol), [(5S) -5-amino-5,6-dihydro-2H-pyran-2-yl] methyl A mixture of acetate (129 mg, 0.754 mmol) and N, N-diisopropylethylamine (0.26 mL, 1.5 mmol) was heated at 90 ° C. for 2 hours. The reaction mixture was concentrated and purified by flash chromatography to give the desired product (0.21 g, 83%). _{C 15 H 16 N 3 O 5} S (M + H) + for calculated LCMS: m / z = 350.1; Found: 350.0.

Step 8. {(5S) -5-[(6-Aminothieno [3,2-b] pyridin-7-yl) amino] tetrahydro-2H-pyran-2-yl} methylacetate {(5S in methanol (4.0 mL) ) -5-[(6-Nitrothieno [3,2-b] pyridin-7-yl) amino] -5,6-dihydro-2H-pyran-2-yl} methyl acetate (210 mg, 0.600 mmol) and carbon (0.21 g) Over 10% palladium mixture was subjected to H ₂ balloon pressure for 2 hours at room temperature. The mixture was filtered and the filtrate was concentrated and purified by flash chromatography (eluting with 15% methanol in dichloromethane) to give the desired product (145 mg, 75%). _{C 15 H 20 N 3 O 3} S (M + H) + for calculated LCMS: m / z = 322.1; Found: 322.0.

Step 9. (1R) -1- {1-[(3S) -6- (hydroxymethyl) tetrahydro-2H-pyran-3-yl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridine 2-yl} ethanol A mixture of (2R) -2-hydroxypropanamide (131 mg, 1.47 mmol) and triethyloxonium tetrafluoroborate (263 mg, 1.38 mmol) in THF (2 mL) at room temperature for 2 hours. Stir. The solvent was removed and the residue was dissolved in ethanol (0.85 mL) and {(5S) -5-[(6-aminothieno [3,2-b] pyridine- in ethanol (3.1 mL) 7-yl) amino] tetrahydro-2H-pyran-2-yl} methyl acetate (145 mg, 0.451 mmol) was added to the suspension. The mixture was stirred at 80 ° C. for 1 hour. The reaction was cooled to room temperature and diluted with water (1.0 mL). Lithium hydroxide (32.4 mg, 1.35 mmol) was added and the mixture was stirred for 2 hours. The reaction mixture is diluted with methanol and purified by preparative LCMS (XBridge C18 column, eluted with a gradient of acetonitrile / water containing 0.1% ammonium hydroxide at a flow rate of 60 mL / min) to give the desired product. The product was obtained as a white solid (95 mg, 63%). _{C 16 H 20 N 3 O 3} S (M + H) + for calculated LCMS: m / z = 334.1; Found: 334.0.

Step 10: ((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridin-1-yl} Tetrahydro-2H-pyran-2-yl) methyl 4-methylbenzenesulfonate and ((2S, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] Thieno [3,2-b] pyridin-1-yl} tetrahydro-2H-pyran-2-yl) methyl 4-methylbenzenesulfonate in methylene chloride (3.4 mL) and pyridine (0.146 mL, 1.80 mmol) (1R) -1- {1-[(3S) -6- (hydroxymethyl) tetrahydro-2H-pyran-3-yl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridine -2-yl } To a solution of ethanol (100 mg, 0.300 mmol) (previous step), p-toluenesulfonyl chloride (57.2 mg, 0.300 mmol) and 4-dimethylaminopyridine (1.8 mg, 0.015 mmol) were added. Added at 0 ° C. The mixture was slowly warmed to room temperature overnight. The reaction mixture was concentrated, diluted with methanol and purified by preparative LCMS (XBridge C18 column, eluted with a gradient of acetonitrile / water containing 0.1% ammonium hydroxide at a flow rate of 60 mL / min). Two peaks were obtained. Analytical HPLC (Waters SunFire C18, 2.1 × 50 mm, 5 μM; flow rate 3 mL / min; injection volume 2 μL; 3 min with 2-80% B gradient (A = water with 0.025% TFA, B = Acetonitrile)): first peak (45.3 mg, 31%) retention time 1.81 min, LCMS calculated for C ₂₃ H ₂₆ N ₃ O ₅ S ₂ (M + H) ⁺ : m / z = 488.1; Measurement: 488.1. LCMS calculated for second peak (8.5 mg, 5.8%) retention time 1.88 min, C ₂₃ H ₂₆ N ₃ O ₅ S ₂ (M + H) ⁺ : m / z = 488.1; found : 488.1.

Step 11. ((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridin-1-yl} tetrahydro-2H -(Pyran-2-yl) acetonitrile ((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] in dimethyl sulfoxide (0.4 mL) Thieno [3,2-b] pyridin-1-yl} tetrahydro-2H-pyran-2-yl) methyl 4-methylbenzenesulfonate (from the first peak of the previous step, 27 mg, 0.055 mmol) and sodium cyanide A mixture of (4.5 mg, 0.092 mmol) was stirred at 50 ° C. for 4 hours. After cooling, the mixture was diluted with methanol and purified by preparative LCMS (XBridge C18 column, eluted with a gradient of acetonitrile / water containing 0.1% ammonium hydroxide at a flow rate of 30 mL / min) and desired. Of 14.5 mg (76%) was obtained. _{C 17 H 19 N 4 O 2} S (M + H) + for calculated LCMS: m / z = 343.1; Found: 343.0. ¹ H NMR (DMSO-d ₆ , 500 MHz) δ9.51 (1H, s), 8.45 (1H, d, J = 5.5 Hz), 7.97 (1H, d, J = 5.5 Hz), 5.31 (1H, m), 5.20 (1H, m), 4.31 (1H, m), 4.23 (1H, m), 4.02 (1H, m), 2.96 (1H) , Dd, J = 17.0 and 4.5 Hz), 2.85 (1H, dd, J = 17.0 and 4.5 Hz), 2.66 (1H, m), 2.26 (1H, m) 2.09 (1H, m), 1.73 (1H, m), 1.69 (3H, d, J = 6.5 Hz) ppm.

実施例２５からの（（２Ｒ，５Ｓ）−５−｛２−［（１Ｒ）−１−ヒドロキシエチル］−１Ｈ−イミダゾ［４，５−ｄ］チエノ［３，２−ｂ］ピリジン−１−イル｝テトラヒドロ−２Ｈ−ピラン−２−イル）アセトニトリル（５２ｍｇ、０．１５ｍｍｏｌ）を、アセトニトリル（８ｍＬ）と水（４ｍＬ）の混合物から結晶化させた。捕集され得られた無色のプリズム結晶は、Ｘ線結晶構造解析に適していた。 Example J1a. ((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridin-1-yl} tetrahydro-2H -Pyran-2-yl) acetonitrile hydrate

((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridine-1- from Example 25 IL} tetrahydro-2H-pyran-2-yl) acetonitrile (52 mg, 0.15 mmol) was crystallized from a mixture of acetonitrile (8 mL) and water (4 mL). The colorless prism crystals collected were suitable for X-ray crystal structure analysis.

Crystal data are: ~ 0.520 × 0.180 × 0.100 mm, orthorhombic, P212121, a = 6.962 (3) Å, b = 11.531 (4) Å, c = 20.799 (7) Å , Volume = 1669.6 (10) Å ³ , Z = 4, T = −100. ° C., molecular weight = 359.42, density = 1.430 g / cm ³ , μ (Mo) = 0.22 mm ⁻¹ .

  Bruker SMART APEX-II CCD system, MoKalpha radiation, standard focus tube, anode power = 50 kV × 42 mA, crystal to plate distance = 5.0 cm, 512 × 512 pixels / frame, beam center = (256.13,253 .14), total number of frames = 1115, vibration / frame = 0.50 °, exposure / frame = 10.1 seconds / frame, SAINT integration, hkl min / max = (− 9, 9, −15, 15, −27 27), data input to shelx = 17025, specific data = 3975, 2 theta range = 3.92 to 55.72 °, completeness for 2 theta 55.72 = 99.80%, R (int-xl) = 0.0681, SADABS correction applied.

The structure was analyzed using XS (Shelxtl) and refined using the chelxtl software package. Refinement by full-matrix least-squares based on F ^2. Int. Tab. Scatter factors from Vol C Table 4.2.6.8 and 6.1.1.4, number of data = 3975, number of limits = 0, number of parameters = 235, data / parameter ratio = 16.91, F ² Goodness of fit = 1.04, R index [I> 4 Sigma (I)] R1 = 0.0505, wR2 = 0.1242, R index (all data) R1 = 0.0769, wR2 = 0.1401, maximum remaining Difference peak and hole = 0.724 and −0.277 e / Å ³ , refined Flack parameter = −0.12 (13). All of the CH hydrogen atoms were refined using a riding model. OH hydrogen was found from the difference map and fully refined.

  The results showed that this asymmetric unit contained one molecule and one water molecule, as shown by the thermal ellipsoid at a 50% probability level. The stereochemistry at each of the three stereocenters (as indicated in the above compound names and structures) was confirmed. The flack parameter has been refined to 0.28 (24), suggesting an accurate enantiomeric setting.

工程１：２，４，５−トリフルオロ−Ｎ−［（１Ｓ）−２，２，２−トリフルオロ−１−メチルエチル］ベンズアミド
アセトニトリル（５０ｍＬ）中２，４，５−トリフルオロ安息香酸（５．００ｇ、２８．４ｍｍｏｌ）溶液に対して、Ｎ，Ｎ−ジメチルホルムアミド（４０μＬ）、続いて塩化オキサリル（３．６０ｍＬ、４２．６ｍｍｏｌ）を加えた。９０分後に、減圧下で揮発性物質を除去した。残留物をアセトニトリル（５０ｍＬ）と一緒に同時蒸発させた。次いで、残留物を塩化メチレン（５０ｍＬ）中に溶かした。この溶液を、トルエン（１００ｍＬ）中（２Ｓ）−１，１，１−トリフルオロプロパン−２−アミン塩酸塩（５．５２ｇ、３６．９ｍｍｏｌ）（Ｓｙｎｑｕｅｓｔ製、９８％ ｅｅ）と、０．５Ｍ水酸化ナトリウム水溶液（１４２ｍＬ、７１．０ｍｍｏｌ）との氷冷された（氷浴）混合物に滴加した。添加後、氷浴を取り除き、反応を室温まで温めた。反応を一晩撹拌した。有機層を分離した。水層は、塩化メチレン（５０ｍＬ）で抽出した。混合した有機層を、２０％塩水（７５ｍＬ）及び水（２×７５ｍＬ）で洗浄し、ＭｇＳＯ_４で乾燥させ、濾過し、減圧下で濃縮して、更なる精製無しに次の工程で直接使用された所望の生成物（６．４９ｇ、８４％）を得た。^１Ｈ ＮＭＲ（３００ＭＨｚ，ＤＭＳＯ−ｄ_６）δ９．０１（ｄ，Ｊ＝７．６Ｈｚ，１Ｈ），７．９２−７．５０（ｍ，２Ｈ），４．７６（ｍ，１Ｈ），１．３１（ｄ，Ｊ＝７．０Ｈｚ，３Ｈ）ｐｐｍ。Ｃ_１０Ｈ_８Ｆ_６ＮＯ（Ｍ＋１）^＋について計算されたＬＣＭＳ：ｍ／ｚ＝２７２．０；測定値：２７２．０。 Example J2.4- [3- (cyanomethyl) -3- (3 ', 5'-dimethyl-1H, 1'H-4,4'-bipyrazol-1-yl) azetidin-1-yl] -2, 5-Difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide

Step 1: 2,4,5-trifluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide 2,4,5-trifluorobenzoic acid in acetonitrile (50 mL) ( 5.00 g, 28.4 mmol) solution was added N, N-dimethylformamide (40 μL) followed by oxalyl chloride (3.60 mL, 42.6 mmol). After 90 minutes, volatiles were removed under reduced pressure. The residue was coevaporated with acetonitrile (50 mL). The residue was then dissolved in methylene chloride (50 mL). This solution was added (2S) -1,1,1-trifluoropropan-2-amine hydrochloride (5.52 g, 36.9 mmol) (from Synquest, 98% ee) in toluene (100 mL) to 0.5 M. To an ice-cold (ice bath) mixture with aqueous sodium hydroxide (142 mL, 71.0 mmol) was added dropwise. After the addition, the ice bath was removed and the reaction was allowed to warm to room temperature. The reaction was stirred overnight. The organic layer was separated. The aqueous layer was extracted with methylene chloride (50 mL). The combined organic layers are washed with 20% brine (75 mL) and water (2 × 75 mL), dried over MgSO ₄ , filtered, concentrated under reduced pressure and used directly in the next step without further purification. The desired product was obtained (6.49 g, 84%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 9.01 (d, J = 7.6 Hz, 1H), 7.92-7.50 (m, 2H), 4.76 (m, 1H), 1. 31 (d, J = 7.0 Hz, 3H) ppm. _{C 10 H 8 F 6 NO (} M + 1) + The calculated LCMS: m / z = 272.0; Found: 272.0.

Step 2: 2,5-Difluoro-4- (3-hydroxyazetidin-1-yl) -N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide in acetonitrile (25 mL) Of 2,4,5-trifluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide (6.39 g, 23.6 mmol), azetidin-3-ol hydrochloride ( 3.19 g, 28.3 mmol) and 1,8-diazabicyclo [5.4.0] undec-7-ene (8.81 mL, 58.9 mmol) were stirred at 80 ° C. for 2 hours. The reaction mixture was diluted with EtOAc (75 mL) and washed with 1N HCl (50 mL), 1N NaHCO ₃ (60 mL), 20% brine (50 mL) and water (75 mL). The aqueous layer was extracted with EtOAc (100 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated under reduced pressure to give the desired product (7.59 g, 91.8%). ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.38 (dd, J = 8.9, 1.9 Hz, 1H), 7.27 (dd, J = 12.8, 6.5 Hz, 1H), 6 .38 (dd, J = 12.3, 7.5 Hz, 1H), 5.71 (d, J = 6.4 Hz, 1H), 4.74 (dp, J = 15.3, 7.6 Hz, 1H) ), 4.62-4.46 (m, 1H), 4.30-4.15 (m, 2H), 3.71 (m, 2H), 1.29 (d, J = 7.1 Hz, 3H) ) Ppm. _{C 13 H 14 F 5 N 2} O 2 (M + 1) + The calculated LCMS: m / z = 325.1; Found: 325.1.

Step 3: 2,5-Difluoro-4- (3-oxoazetidin-1-yl) -N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide 2 in methylene chloride (93 mL) , 5-Difluoro-4- (3-hydroxyazetidin-1-yl) -N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide (7.57 g, 23.3 mmol) To the solution at room temperature, iodobenzene diacetate (9.40 g, 29.2 mmol) and 2,2,6,6-tetramethyl-1-piperidinyloxy free radical (1.82 g, 11.7 mmol) ( TEMPO) was added. The reaction mixture was stirred at room temperature overnight. The mixture was diluted with EtOAc (100 mL) and washed with 0.5 N NaHCO ₃ (2 × 80 mL), 20% brine (100 mL) and water (100 mL). The aqueous layer was extracted with ethyl acetate (75 mL). The organic layers were combined, dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography using a silica gel column eluting with 0% to 5% ethyl acetate in methylene chloride to give the crude product and recrystallized from MTBE (50 mL) and heptane (100 mL). To give the desired product (5.44 g, 72%) as a colorless solid. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 8.52 (d, J = 8.0 Hz, 1H), 7.36 (dd, J = 12.5, 6.5 Hz, 1H), 6.63 (dd , J = 12.1, 7.6 Hz, 1H), 4.90 (d, J = 2.1 Hz, 4H), 4.86-4.68 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H) ppm. _{C 13 H 12 F 5 N 2} O 2 (M + 1) + The calculated LCMS: m / z = 323.1; Found: 323.0.

Step 4: 4- [3- (Cyanomethylene) azetidin-1-yl] -2,5-difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide diethylcyanomethyl Phosphonate (1.95 mL, 11.8 mmol) is added dropwise to a cooled (ice bath) solution of 1.0 M potassium tert-butoxide in THF (11.8 mL, 11.8 mmol) diluted with tetrahydrofuran (12 mL). did. The ice bath was removed and the reaction was warmed to room temperature and stirred for 90 minutes. The reaction solution was cooled again with an ice bath. The above prepared solution was then added to 2,5-difluoro-4- (3-oxoazetidin-1-yl) -N-[(1S) -2,2,2-trifluoro-1-methyl in tetrahydrofuran (50 mL). Ethyl] benzamide (4.00 g, 12.4 mmol) was added over 12 minutes to a cooled (ice bath) solution. The reaction mixture was stirred for 30 minutes. The ice bath was removed and the reaction was stirred at room temperature overnight and then quenched by adding 20% brine (75 mL) and ethyl acetate (75 mL). The organic layer was separated. The aqueous layer was extracted with ethyl acetate (50 mL). The combined organic layers were dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash chromatography on a silica gel column with ethyl acetate in hexane (0-30%) to give the desired product (2.6 g). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.59-8.37 (m, 1H), 7.33 (dd, J = 12.5, 6.4 Hz, 1H), 6.59 (dd, J = 12.0, 7.4 Hz, 1H), 5.88 (m, 1H), 4.94-4.75 (m, 4H), 4.76 (m, 1H), 1.31 (d, J = 7.1 Hz, 3H) ppm. _{C 15 H 13 F 5 N 3} O (M + 1) + The calculated LCMS: m / z = 346.1; Found: 346.1.

Step 5: 4- {3- (cyanomethyl) -3- [4- (4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl) -1H-pyrazol-1-yl] Azetidin-1-yl} -2,5-difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide 4- (4,4,4) in acetonitrile (20.2 mL) 5,5-Tetramethyl-1,3,2-dioxaboron-2-yl) -1H-pyrazole (1.00 g, 5.15 mmol), 4- [3- (cyanomethylene) azetidin-1-yl] -2 , 5-Difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide (1.78 g, 5.15 mmol), and 1,8-diazabicyclo [5.4.0] Undec-7-ene (0.31 (mL, 2.1 mmol) was heated at 50 ° C. overnight. After cooling, the solvent was removed under reduced pressure. The residue was used in the next step without further purification. _{C 24 H 28 BF 5 N 5} O 3 (M + 1) + The calculated LCMS: m / z = 540.2; Found: 540.1.

Step 6: 4- [3- (Cyanomethyl) -3- (3 ′, 5′-dimethyl-1H, 1′H-4,4′-bipyrazol-1-yl) azetidin-1-yl] -2,5 -Difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide 4- {3- (cyanomethyl) -3 in 1,4-dioxane (10 mL) / water (5 mL) -[4- (4,4,5,5-tetramethyl-1,3,2-dioxaboron-2-yl) -1H-pyrazol-1-yl] azetidin-1-yl} -2,5-difluoro- N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide (329 mg, 0.610 mmol), 4-bromo-3,5-dimethyl-1H-pyrazole (206 mg, 1.18 mmol) , Tetrakis (triphenylphos Fin) palladium (0) (110 mg, 0.098 mmol), and the mixture of sodium carbonate (320 mg, 3.0 mmol), purged with nitrogen, and stirred for 1 hour at 110 ° C.. The reaction mixture was diluted with EtOAc, washed with water and brine, and concentrated. The residue was first purified on silica gel (eluting with 0-100% EtOAc / hexanes, followed by 10% methanol / dichloromethane), then preparative LCMS (XBridge C18 column, 60 mL / min flow rate, Elution with a gradient of acetonitrile / water containing 0.1% ammonium hydroxide) to give the desired product (30 mg, 9.7%). ¹ H NMR (500 MHz, DMSO-d ₆ ) δ 12.17 (1 H, s), 8.45 (1 H, d, J = 8.0 Hz), 8.10 (1 H, s), 7.70 (1 H, s), 7.34 (1H, m), 6.61 (1H, s), 4.77 (1H, m), 4.62 (2H, d, J = 9.0 Hz), 4.39 (1H) , D, J = 9.0 Hz), 3.64 (2H, s), 2.22 (6H, s), 1.31 (6H, d, J = 7.0 Hz) ppm. _{C 23 H 23 F 5 N 7} O (M + H) + for calculated LCMS: m / z = 508.2; Found: 508.0.

Example A: In Vitro JAK Kinase Assay Compounds of formula I herein are prepared according to Park et al. The inhibitory activity of the JAK target was tested according to the following in vitro assay described in, Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (aa 837-1142), JAK2 (aa 828-1132) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1 and JAK2 was assayed by measuring the phosphorylation of biotinylated peptides. The phosphorylated peptide was detected by homogeneous time-resolved fluorescence (HTRF). Compound IC ₅₀ contains enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg / mL (0.01%) BSA. Measured for each kinase in a 40 μL reaction. With a 1 mM IC ₅₀ measurement, the ATP concentration in the reaction was 1 mM. The reaction was performed at room temperature for 1 hour and then stopped with 20 μL of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the europium labeled antibody occurred for 40 minutes and the HTRF signal was read with a Fusion plate reader (Perkin Elmer, Boston, Mass.).

Example B: Cellular Assay A cancer cell line that relies on JAK / STAT signaling for growth was grown at 6000 cells / well (96 cells in RPMI 1640, 10% FBS, and 1 nG / mL of the appropriate cytokine. (Well plate type). Compounds can be added to cells in DMSO / medium (final concentration 0.2% DMSO) and incubated for 72 hours at 37 ° C., 5% CO ₂ . The effect of the compound on cell survival is assessed using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) followed by TopCount (Perkin Elmer, Boston, Mass.) Quantification. Potential off-target effects of compounds are measured in parallel using non-JAK derived cell lines with the same assay measurements. All experiments are typically repeated.

  The cell lines described above can also be used to examine the effects of compounds on phosphorylation of JAK kinases or potential downstream substrates such as STAT protein, Akt, Shp2, or Erk. These experiments can be performed after overnight cytokine deprivation treatment, followed by a brief pre-incubation with the compound (2 hours or less) and cytokine stimulation of about 1 hour or less. The protein is then extracted from the cells and analyzed by techniques well known to those skilled in the art, including Western blotting or ELISA, using antibodies that can distinguish between phosphorylated and total proteins. These experiments can utilize normal cells or cancer cells to examine the activity of compounds related to tumor cell survival biology or mediators of inflammatory diseases. For example, with respect to the latter, cytokines such as IL-6, IL-12, IL-23, or IFN can be used to stimulate JAK activation to phosphorylate STAT protein (s) and potentially , Transcriptional profiles of proteins such as IL-17 (assessed by array or qPCR techniques) or production and / or secretion. The ability of a compound to inhibit these cytokine-mediated effects can be measured using techniques common to those skilled in the art.

  The compounds described herein can also be tested in cell models designed to assess their potency and activity against mutant JAKs, such as the JAK2V617F mutation found in myeloproliferative disorders. These experiments often utilize hematological lineage cytokine-dependent cells (eg, BaF / 3), in which wild-type or mutant JAK kinases are expressed ectopically (James, C. et al. , Et al. Nature 434: 1144-1148; Staerk, J., et al. JBC 280: 41893-41899). Endpoints include cell survival, proliferation, and the effects of compounds on phosphorylated JAK, STAT, Akt, or Erk proteins.

Certain compounds described herein can be evaluated for their activity to inhibit T cell proliferation. Such an assay can be considered a second cytokine (ie JAK) driven proliferation assay and also a simple assay of immunosuppression or inhibition of immune activation. The following is a brief overview on how such an experiment can be performed. Peripheral blood mononuclear cells (PBMC) can be prepared from human whole blood samples using the Ficoll Hyperpaque separation method and T cells (fraction 2000) can be obtained from PBMCs by chickenpox. Freshly isolated human T cells were cultured in RPMI supplemented with medium (10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin at a density of 2 × 10 ⁶ cells / ml at 37 ° C. for up to 2 days. 1640). For cell proliferation analysis stimulated with IL-2, T cells were first treated with phytohemagglutin (PHA) at a final concentration of 10 μg / mL for 72 hours. After washing once with PBS, 6000 cells / well are seeded in a 96-well plate and treated with different concentrations of compounds in the medium in the presence of 100 U / mL human IL-2 (ProSpec-Tany TechnoGene; Rehovot, Israel) did. The plates are incubated at 37 ° C. for 72 hours and the growth index is assessed using CellTiter-Glo Luminescent reagent according to the manufacturer's proposed protocol (Promega; Madison, Wis.).

Example C: In vivo anti-tumor effect The compounds described herein can be evaluated in a human tumor xenograft model in immunocompromised mice. For example, tumor-forming mutants of the INA-6 plasmacytoma cell line can be used to inoculate SCID mice subcutaneously (Burger, R., et al. Hematol J. 2: 42-53, 2001). . The tumorigenic animals can then be randomized into drug or vehicle treatment groups, and different numbers of compounds can be administered in any number of conventional routes, including continuous infusion using oral, intraperitoneal, or implantable pumps. Can be administered. Tumor growth is followed over time using a caliper. In addition, tumor samples can be collected at any time after the start of the above (Example B) analysis process to assess the effects of compounds on JAK activity and downstream signaling pathways. In addition, the selectivity of the compound (s) can be assessed using a xenograft tumor model caused by other known kinases (eg Bcr-Abl), such as the K562 tumor model.

Example D: Mouse Skin Contact Delayed Hypersensitivity Response Test Compounds described herein can also be tested for their effects (inhibiting JAK targets) in a mouse delayed hypersensitivity test model caused by T cells. . Mouse skin contact delayed hypersensitivity (DTH) response is considered to be an effective model of clinical contact dermatitis and other T lymphocyte-mediated immune disorders of the skin, such as psoriasis (Immunol Today. 1998 Jan; 19 (1): 37-44). Mouse DTH shares multiple features with psoriasis, such as immune infiltration, concomitant increase in inflammatory cytokines, and keratinocyte hyperproliferation. Furthermore, many classes of drugs that are effective in treating psoriasis in the clinic are also effective inhibitors of the DTH response in mice (Agents Actions. 1993 Jan; 38 (1-2): 116-21). .

  On days 0 and 1, Balb / c mice were sensitized with topical application to their shaved abdomen with the antigen 2,4, dinitro-fluorobenzene (DNFB). On day 5, the ear is measured for thickness using a technician micrometer. Record this measurement and use it as a baseline. Both animal ears were then challenged by topical application of DNFB at a concentration of 0.2% in a total volume of 20 μL (10 μL inside the auricle and 10 μL outside the auricle). Ears are measured again 24-72 hours after challenge. Treatment with the test compound is administered through or prior to the sensitization and challenge phase (1-7 days) and through the challenge phase (usually 4 pm-7 days). Treatment of the test compound (at different concentrations) is administered systemically or locally (local application of treatment to the ear). The effectiveness of the test compound is indicated by a reduction in ear swelling compared to the untreated situation. Compounds that produced a reduction of 20% or more were considered effective. In some experiments, mice are challenged but not sensitized (negative control).

  The inhibitory effect of the test compound (inhibiting the activation of the JAK-STAT pathway) can be confirmed by immunohistochemical analysis. Activation of the JAK-STAT pathway (s) results in the formation and translocation of functional transcription factors. Furthermore, the influx of immune cells and increased proliferation of keratinocytes should also provide a change in the unique expression profile in the ear that can be examined and quantified. Formalin-fixed, paraffin-embedded ear fragments (collected after the challenge step in the DTH model) were immunized with antibodies that specifically interact with phosphorylated STAT3 (clone 58E12, Cell Signaling Technologies). Analyze histochemically. Mouse ears are treated with a test compound, vehicle, or dexamethasone (clinically effective treatment for psoriasis) or no treatment in the DTH model for comparison. Test compounds and dexamethasone can produce similar transcriptional changes, both qualitatively and quantitatively, and both the test compound and dexamethasone can reduce the number of infiltrating cells. Both systemic and local administration of the test compound can lead to an inhibitory effect, ie a reduction in the number of infiltrating cells and inhibition of transcriptional changes.

Example E: In vivo anti-inflammatory activity The compounds described herein can be evaluated in rodents or non-rodents designed to reproduce a single or multiple inflammatory response . For example, a rodent animal model of arthritis can be used to assess the therapeutic effect of a compound administered prophylactically or therapeutically. These models include, but are not limited to, mouse or rat collagen-induced arthritis, rat adjuvant-induced arthritis, and collagen antibody-induced arthritis. Include, but are not limited to, multiple sclerosis, type I diabetes, uveoretinitis, thyroiditis, myasthenia gravis, immunoglobulin nephropathy, myocarditis, airway sensitization (asthma), lupus, or colitis Non-autoimmune diseases may also be used to assess the therapeutic effects of the compounds described herein. These models are well established in research institutions and are known to those skilled in the art (Current Protocols in Immunology, Vol 3., Coligan, JE et al, Wiley Press .; Methods in Molecular Biology: 225, Inflammation Protocols, Winyard, PG and Willoughby, DA, Humana Press, 2003.).

  Each journal or patent document is hereby incorporated by reference in its entirety.

Claims (54)

  A method of prolonging survival or progression-free survival in a patient with a solid tumor, said patient having an elevated serum concentration of C-reactive protein (CRP), wherein said patient has a JAK inhibitor or IL -6 said method comprising administering a signaling inhibitor, said administration extending the survival or progression free survival of the patient.   2. The method of claim 1, wherein the method further comprises selecting a patient having an elevated serum concentration of C-reactive protein prior to the administration. (A) selecting a solid tumor patient having a serum concentration of CRP that is greater than or equal to the median of the baseline serum concentration of C-reactive protein (CRP) for a patient population having a solid tumor;
(B) A method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor. (A) selecting said solid tumor patient having a serum concentration of C-reactive protein (CRP) that is about 10 μg / mL or greater;
(B) A method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.   5. The method of claim 3 or 4, wherein the administration extends the survival time of the patient.   The method according to claim 3 or 4, wherein the administration prolongs progression free survival of the patient.   The method according to claim 1, wherein the serum concentration of CRP is about 13 μg / mL or more.   The patient has an improved Glasgow prognostic score (mGPS) of 1 or 2, and the patient comprises administering a Janus kinase (JAK) inhibitor or an IL-6 signaling inhibitor to the patient. A method of treating a solid tumor in a patient in need. (A) selecting patients with solid tumors with an improved Glasgow prognostic score of 1 or 2;
(B) A method of treating a solid tumor comprising administering to the patient a therapeutically effective amount of a JAK inhibitor or an IL-6 signaling inhibitor.   10. The method of claim 9, wherein the administration extends a patient's survival time.   10. The method of claim 9, wherein the administration extends progression free survival of the patient.   The solid tumor is prostate cancer, renal cancer, liver cancer, colon cancer, rectal cancer, renal cancer, colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, Kaposi sarcoma, The method according to any one of claims 1 to 11, which is melanoma, esophageal cancer, gastroesophageal cancer, cervical cancer, hepatocellular carcinoma, endometrial cancer, urothelial cancer, or ovarian cancer.   The method according to claim 1, wherein the solid tumor is prostate cancer, pancreatic cancer, stomach cancer, colon cancer, or lung cancer.   The method according to claim 1, wherein the solid tumor is pancreatic cancer.   The method according to claim 1, wherein the solid tumor is endometrial cancer.   The method according to claim 1, wherein the solid tumor is non-small cell lung cancer.   The method according to any one of claims 1 to 16, wherein the method comprises administering a JAK inhibitor to the patient.   17. The method of any one of claims 1-16, wherein the method comprises administering an IL-6 signaling inhibitor to the patient.   The method according to any one of claims 1 to 18, wherein the JAK inhibitor is ruxolitinib or a pharmaceutically acceptable salt thereof.   20. The method of any one of claims 1-19, wherein the method comprises administering to the patient about 15 mg to about 25 mg BID ruxolitinib or a pharmaceutically acceptable salt thereof based on the free base. the method of.   21. The method of any one of claims 1-20, wherein the method further comprises administering one or more additional chemotherapeutic agents to the patient.   23. The method of claim 21, wherein the one or more chemotherapeutic agents are selected from antimetabolites, topoisomerase 1 inhibitors, platinum analogs, taxanes, and anthracyclines, EGFR inhibitors, and combinations thereof.   The one or more additional chemotherapeutic agents include capecitabine, gemcitabine, Abraxane® (paclitaxel protein-binding particles for injectable suspension), docetaxel, fluorouracil (5-FU), oxaliplatin, cisplatin, 24. The method of claim 21, wherein the method is selected from carboplatin, irinotecan, topotecan, paclitaxel, leucovorin, doxorubicin, and combinations thereof.   24. The method of claim 21, wherein the one or more additional chemotherapeutic agents is capecitabine.   A method for predicting the effectiveness of a treatment using a JAK inhibitor or an IL-6 signaling inhibitor for a patient having a solid tumor, wherein the serum concentration of the C-reactive protein (CRP) in the patient is determined. Comparing to the baseline serum concentration of CRP of the patient population having the solid tumor, wherein the serum CRP concentration in the patient above the baseline serum concentration is the JAK inhibitor or IL-6 signaling inhibition The method, wherein the method represents the effectiveness of a treatment using an agent on the patient.   26. The method of claim 25, wherein the method further comprises measuring a serum concentration of the patient's CRP using a CRP assay prior to the comparison.   27. The method of claim 25 or 26, wherein the method further comprises formulating a JAK inhibitor or an IL-6 signaling inhibitor for the patient.   28. A method according to any one of claims 25 to 27, wherein the efficacy is an improvement in the overall survival of the patient.   28. A method according to any one of claims 25 to 27, wherein the efficacy is an improvement in progression free survival of the patient. (A) selecting patients with solid tumors with an improved Glasgow prognostic score of 1 or 2;
(B) administering to the patient a therapeutically effective amount of an inhibitor of ruxolitinib or a pharmaceutically acceptable salt thereof, wherein the treatment results in an extension of the patient's survival or progression free survival, A method of treating a solid tumor.   32. The method of claim 30, wherein the method further comprises administering one or more additional chemotherapeutic agents to the patient.   32. The method of claim 31, wherein the one or more chemotherapeutic agents are selected from antimetabolites, topoisomerase 1 inhibitors, platinum analogs, taxanes, and anthracyclines, EGFR inhibitors, and combinations thereof.   The one or more additional chemotherapeutic agents include capecitabine, gemcitabine, Abraxane® (paclitaxel protein-binding particles for injectable suspension), docetaxel, fluorouracil (5-FU), oxaliplatin, cisplatin, 32. The method of claim 31, wherein the method is selected from carboplatin, irinotecan, topotecan, paclitaxel, leucovorin, doxorubicin, and combinations thereof.   32. The method of claim 31, wherein the one or more additional chemotherapeutic agents is capecitabine.   The solid tumor is prostate cancer, renal cancer, liver cancer, colon cancer, rectal cancer, renal cancer, colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, head and neck cancer, thyroid cancer, glioblastoma, Kaposi sarcoma, The method according to any one of claims 30 to 34, which is melanoma, esophageal cancer, gastroesophageal cancer, cervical cancer, hepatocellular carcinoma, endometrial cancer, urothelial cancer, or ovarian cancer.   35. The method according to any one of claims 30 to 34, wherein the solid tumor is prostate cancer, pancreatic cancer, stomach cancer, colon cancer, or lung cancer.   The method according to any one of claims 30 to 34, wherein the solid tumor is pancreatic cancer.   The method according to any one of claims 30 to 34, wherein the solid tumor is endometrial cancer.   The method according to any one of claims 30 to 34, wherein the solid tumor is non-small cell lung cancer.   A method for predicting the efficacy of a treatment using ruxolitinib or a pharmaceutically acceptable salt thereof for a pancreatic cancer patient, wherein the patient's serum concentration of C-reactive protein (CRP) is determined by the patient having the solid tumor Treatment wherein the serum CRP concentration in the patient above the baseline serum concentration is using an inhibitor of ruxolitinib or a pharmaceutically acceptable salt thereof, comprising comparing to a baseline serum concentration of CRP in a population Wherein said method represents efficacy for said patient.   41. The method of claim 40, wherein the method further comprises administering a therapeutically effective amount of an additional chemotherapeutic agent to the patient.   41. The method of claim 40, wherein the one or more chemotherapeutic agents are selected from antimetabolites, topoisomerase 1 inhibitors, platinum analogs, taxanes, and anthracyclines, EGFR inhibitors, and combinations thereof.   The one or more additional chemotherapeutic agents include capecitabine, gemcitabine, Abraxane® (paclitaxel protein-binding particles for injectable suspension), docetaxel, fluorouracil (5-FU), oxaliplatin, cisplatin, 41. The method of claim 40, selected from carboplatin, irinotecan, topotecan, paclitaxel, leucovorin, doxorubicin, and combinations thereof.   41. The method of claim 40, wherein the one or more additional chemotherapeutic agents are capecitabine.   45. A method according to any one of claims 40 to 44, wherein the efficacy is an improvement in the survival of the patient.   45. The method of any one of claims 40 to 44, wherein the efficacy is an improvement in progression free survival of the patient.   The patient has an improved Glasgow prognostic score (mGPS) of 1 or 2, and the patient comprises administering a Janus kinase (JAK) inhibitor or an IL-6 signaling inhibitor to the patient. A method of treating diffuse large B-cell lymphoma in a patient in need thereof.   The method according to any one of claims 1 to 47, wherein the JAK inhibitor is a selective JAK1 inhibitor. The selective JAK1 inhibitor is:
3- [1- (6-Chloropyridin-2-yl) pyrrolidin-3-yl] -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1 -Yl] propanenitrile;
3- (1- [1,3] oxazolo [5,4-b] pyridin-2-ylpyrrolidin-3-yl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidine-4- Yl) -1H-pyrazol-1-yl] propanenitrile;
4-[(4- {3-cyano-2- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] propyl} piperazin-1-yl) Carbonyl] -3-fluorobenzonitrile;
4-[(4- {3-cyano-2- [3- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrrol-1-yl] propyl} piperazin-1-yl) Carbonyl] -3-fluorobenzonitrile;
{1- {1- [3-Fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl)- 1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile;
4- {3- (cyanomethyl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-1-yl} -N- [ 4-fluoro-2- (trifluoromethyl) phenyl] piperidine-1-carboxamide;
[3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] -1- (1-{[2- (trifluoromethyl) pyrimidine-4 -Yl] carbonyl} piperidin-4-yl) azetidin-3-yl] acetonitrile;
[Trans-1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] -3- (4-{[2- (trifluoromethyl) pyrimidine -4-yl] carbonyl} piperazin-1-yl) cyclobutyl] acetonitrile;
{Trans-3- (4 {[4-[(3-hydroxyazetidin-1-yl) methyl] -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidin-1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
{Trans-3- (4-{[4-{[(2S) -2- (hydroxymethyl) pyrrolidin-1-yl] methyl} -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidine- 1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
{Trans-3- (4-{[4-{[(2R) -2- (hydroxymethyl) pyrrolidin-1-yl] methyl} -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidine- 1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
4- (4- {3-[(dimethylamino) methyl] -5-fluorophenoxy} piperidin-1-yl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] butanenitrile;
5- {3- (cyanomethyl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-1-yl} -N-isopropyl Pyrazine-2-carboxamide;
4- {3- (cyanomethyl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-1-yl} -2,5 -Difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide;
5- {3- (cyanomethyl) -3- [4- (1H-pyrrolo [2,3-b] pyridin-4-yl) -1H-pyrazol-1-yl] azetidin-1-yl} -N-isopropyl Pyrazine-2-carboxamide;
{1- (cis-4-{[6- (2-hydroxyethyl) -2- (trifluoromethyl) pyrimidin-4-yl] oxy} cyclohexyl) -3- [4- (7H-pyrrolo [2,3 -D] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile;
{1- (cis-4-{[4-[(Ethylamino group) methyl] -6- (trifluoromethyl) pyridin-2-yl] oxy} cyclohexyl) -3- [4- (7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile;
{1- (cis-4-{[4- (1hydroxy1-methylethyl) -6- (trifluoromethyl) pyridin-2-yl] oxy} cyclohexyl) -3- [4- (7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile;
{1- (cis-4-{[4-{[(3R) -3-hydroxypyrrolidin-1-yl] methyl} -6- (trifluoromethyl) pyridin-2-yl] oxy} cyclohexyl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile;
{1- (cis-4-{[4-{[(3S) -3-hydroxypyrrolidin-1-yl] methyl} -6- (trifluoromethyl) pyridin-2-yl] oxy} cyclohexyl) -3- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl} acetonitrile;
{Trans-3- (4-{[4-({[(1S) -2-hydroxy-1-methylethyl] amino} methyl) -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidine- 1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
{Trans-3- (4-{[4-({[(2R) -2-hydroxypropyl] amino} methyl) -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidin-1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
{Trans-3- (4-{[4-({[(2S) -2-hydroxypropyl] amino} methyl) -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidin-1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
{Trans-3- (4-{[4- (2-hydroxyethyl) -6- (trifluoromethyl) pyridin-2-yl] oxy} piperidin-1-yl) -1- [4- (7H-pyrrolo [2,3-d] pyrimidin-4-yl) -1H-pyrazol-1-yl] cyclobutyl} acetonitrile;
((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridin-1-yl} tetrahydro-2H -Pyran-2-yl) acetonitrile; and 4- [3- (cyanomethyl) -3- (3 ', 5'-dimethyl-1H, 1'H-4,4'-bipyrazol-1-yl) azetidine-1 -Yl] -2,5-difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide;
49. The method of claim 48, or a pharmaceutically acceptable salt of any of the compounds.   The selective JAK1 inhibitor is ((2R, 5S) -5- {2-[(1R) -1-hydroxyethyl] -1H-imidazo [4,5-d] thieno [3,2-b] pyridine. 49. The method of claim 48, which is -1-yl} tetrahydro-2H-pyran-2-yl) acetonitrile or a pharmaceutically acceptable salt thereof.   The selective JAK1 inhibitor is 4- [3- (cyanomethyl) -3- (3 ′, 5′-dimethyl-1H, 1′H-4,4′-bipyrazol-1-yl) azetidin-1-yl 49. The method of claim 48, which is -2,5-difluoro-N-[(1S) -2,2,2-trifluoro-1-methylethyl] benzamide or a pharmaceutically acceptable salt thereof.   The selective JAK1 inhibitor is {1- {1- [3-fluoro-2- (trifluoromethyl) isonicotinoyl] piperidin-4-yl} -3- [4- (7H-pyrrolo [2,3-d 49. The method of claim 48, which is pyrimidin-4-yl) -1H-pyrazol-1-yl] azetidin-3-yl}) acetonitrile or a pharmaceutically acceptable salt thereof.   48. The method of any one of claims 1-47, further comprising administering one or more non-steroidal anti-inflammatory drugs (NSAIDs) to the patient.   The NSAID is selected from aspirin, diflunisal, salsalate, ibuprofen, naproxen, fenoprofen, ketoprofen, oxaprozin, indomethacin, tolmethine, sulindac, etodolac, ketodolac, piroxicam, meloxicam, aminooxycame, tenoxycam 54. The method of claim 53.

Images